阅读说明：本技术 嵌合分子及其用途 (Chimeric molecule and application thereof ) 是由 K·J·沙佩尔 D·沃特森 P·R·杨 于 2018-03-29 设计创作，主要内容包括：公开了基于病毒膜融合蛋白的嵌合多肽。更具体地,本发明公开了包含病毒融合蛋白的病毒粒表面暴露部分和异源性结构稳定化部分的嵌合多肽和这些嵌合多肽的复合体。本发明还公开了这些复合体在组合物中的用途和用于激发针对包膜病毒融合蛋白或所述融合蛋白复合体的免疫应答的方法和/或用于治疗或预防包膜病毒感染的方法。本发明进一步公开了异源性结构稳定化部分用于寡聚异源目的分子的用途。(Disclose the chimeric polyeptides based on virus membrane-fusion protein.More specifically, the invention discloses the virion surface expose portions comprising virus amalgamation protein and heterologous structural to stabilize the chimeric polyeptides of part and the complex of these chimeric polyeptides.The invention also discloses the purposes of these complexs in the composition and the methods for exciting the method for the immune response for being directed to enveloped virus fusion protein or the fusion protein complex and/or for treating or preventing infection of coated virus.The present invention further discloses heterologous structurals to stabilize the purposes that part is used for the heterologous molecules of interest of oligomerization.)

1. chimeric polyeptides stabilize the enveloped virus that part effectively connects with heterologous structural it includes downstream and merge extracellular structure Domain polypeptide, the heterologous structural stabilize part and include the first complementary heptapeptide repetitive sequence area (HR1) and the second heptapeptide weight The area complex sequences (HR2), the first heptapeptide repetitive sequence area (HR1) of the complementation and second heptapeptide repetitive sequence (HR2) Qu Shi It is associated each other under conditions of (for example, in aqueous solution) their associations to form antiparallel double helix beam.

2. chimeric polyeptides according to claim 1, wherein the area HR1 and the area HR2 lack complementary with extracellular domain polypeptide Property, thus their preferences form antiparallel double helix beam each other, without being formed with the structural member of extracellular domain polypeptide Antiparallel double helix beam.

3. according to claim 1 or chimeric polyeptides as claimed in claim 2, wherein the area HR1 and the area HR2 are each independently with amino The n times of acids type repeat 7 residue patterns and are characterized, and the pattern is expressed as (a-b-c-d-e-f-g-)_nOr (d-e-f-g-a-b- c-)_n, wherein pattern element ' a ' to ' g ' refers to that it is in the conventional septuple position of amino acid classes and n is equal to or greater than 2 Number, and at least 50% (or at least 51% at least 99% and the whole between them of conventional septuple position ' a ' and ' d ' Whole number percentage) occupied by hydrophobic amino acid type and conventional septuple position ' b ', ' c ', ' e ', ' f ' and ' g ' at least 50% (or at least 51% whole whole number percentages at least 99% and between them) are occupied, institute by hydrophilic amino acid type The distribution between hydrophobic amino acid type and hydrophilic amino acid type obtained makes it possible to identify heptapeptide repetitive sequence area.

4. chimeric polyeptides according to any one of claim 1 to 3, wherein one or both includes in the area HR1 and the area HR2 Endogenous I class enveloped virus fusion protein heptapeptide repetitive sequence region amino acid sequence is made from it or consisting essentially of.

5. chimeric polyeptides according to claim 4, wherein the area HR1 and the area HR2 separately include one or more I class coating diseases The complementary endogenous area (HRA) heptapeptide repetitive sequence A and the area heptapeptide repetitive sequence B (HRB) of malicious fusion protein, be made from it or It is consisting essentially of.

6. chimeric polyeptides according to claim 5, wherein HRA region amino acid sequence and HRB region amino acid sequence are derived from Identical I class enveloped virus fusion protein.

7. chimeric polyeptides according to claim 5, wherein HRA region amino acid sequence and HRB region amino acid sequence are derived from Different I class enveloped virus fusion proteins.

8. chimeric polyeptides according to any one of claim 1 to 7, wherein the area HR1 and the area HR2 are independently selected from just viscous disease The area HRA for the fusion protein that poison, paramyxovirus, retrovirus, coronavirus, filamentous form virus and Arenavirus are expressed and HRB Area.

9. chimeric polyeptides according to any one of claim 1 to 8, wherein structure stabilization part (e.g., including HR1 One or both in area and the area HR2) comprising immune silencing moiety, the immune silencing moiety inhibits excitation to be directed to structure stabilization Partial immune response.

10. chimeric polyeptides according to claim 9, wherein immune silencing moiety is by glycosylase (for example, glycosyl shifts Enzyme) identification and glycosylated glycosylation site.

11. chimeric polyeptides according to any one of claim 1 to 10, wherein structure stabilization part (e.g., including One or both in the area HR1 and the area HR2) include one or more unnatural amino acids.

12. chimeric polyeptides according to claim 11, wherein one or more unnatural amino acids allow to be coupled poly- second two Alcohol.

13. chimeric polyeptides according to claim 11, wherein one or more unnatural amino acids allow to be coupled immune thorn Swash part.

14. chimeric polyeptides according to claim 11, wherein one or more unnatural amino acids allow to be coupled lipid (example Such as, to promote to show the formation of the lipid vesicle or virus-like particle of the extracellular domain of chimeric polyeptides, particularly for stimulation of host Immune response).

15. wherein extracellular domain polypeptide corresponds to I class packet according to claim 1 to chimeric polyeptides described in any one of 14 Film virus amalgamation protein extracellular domain.

16. chimeric polyeptides according to claim 15, wherein extracellular domain polypeptide includes the area endogenous HRA and endogenous One or both in the area HRB.

17. according to claim 15 or claim 16 described in chimeric polyeptides, wherein I class fusion protein is from I class coating A kind of fusion protein of fusion protein virus, it is described virus selected from orthomyxovirus, paramyxovirus, retrovirus, coronavirus, Filamentous form virus and Arenavirus.

18. wherein extracellular domain polypeptide corresponds to Group III according to claim 1 to chimeric polyeptides described in any one of 14 Enveloped virus fusion protein extracellular domain.

19. chimeric polyeptides according to claim 18, wherein Group III fusion protein is from Group III envelope fusion protein A kind of fusion protein of virus, the virus are selected from rhabdovirus and herpesviral.

20. according to claim 1 to chimeric polyeptides described in any one of 19, wherein extracellular domain polypeptide (for example, I class or Group III) comprising intact precursor extracellular domain polypeptide or part thereof or by intact precursor extracellular domain polypeptide or part thereof group At.

21. chimeric polyeptides according to claim 20, wherein extracellular domain polypeptide or part lack endogenous signal peptides, Proteolytic cleavage site, the endogenous head point of extracellular domain, the endogenous stem portion of extracellular domain, endogenous mucin Any one in spline structure domain, endogenous film proximal end perimeter and endogenous fusogenic peptide or more persons.

22. according to chimeric polyeptides described in claim 20 or claim 21, wherein change or missing wild type or reference melt One or more endogenous protein cleavage sites (for example, one or more furin cleavage site) of hop protein so that Extracellular domain polypeptide is obtained to be less susceptible to suffer the proteolytic cleavage of protease.

23. wherein extracellular domain polypeptide includes at least one according to claim 1 to chimeric polyeptides described in any one of 22 Epitope before merging, epitope is not present in the fusion of enveloped virus fusion protein corresponding with extracellular domain polypeptide before the fusion Afterwards in form.

24. according to claim 1 to chimeric polyeptides described in any one of 23, the wherein area HR1 of structure stabilization part and HR2 Area is connected by connector.

25. chimeric polyeptides according to claim 24, center tap is by about 1 to about 100 amino acid residue (and they it Between whole integer amino acid residues) composition.

26. chimeric polyeptides according to claim 24, center tap is by about 1 to about 50 amino acid residue (and they it Between whole integer amino acid residues) composition.

27. chimeric polyeptides according to claim 24, center tap by about 50 to about 100 amino acid residues (and they Between whole integer amino acid residues) composition.

28. the chimeric polyeptides according to any one of claim 24 to 27, center tap includes at least one portion, described Part selected from promote the purification part of chimeric polyeptides purifying, metering needle to the immunological regulation part of the immune response of chimeric polyeptides, Cell specific portion and the part for assigning configuration flexibility.

29. chimeric polyeptides, it includes downstreams to stabilize the protein molecule that part is effectively connect with heterologous structural, described heterologous Property structure stabilization part include complementary the first heptapeptide repetitive sequence area (HR1) and second area heptapeptide repetitive sequence (HR2), institute Complementary the first heptapeptide repetitive sequence area (HR1) and the second heptapeptide repetitive sequence area (HR2) are stated in the condition for being suitable for their associations Under (for example, in aqueous solution) associated each other to form antiparallel double helix beam.

30. chimeric polyeptides according to claim 29, wherein protein molecule is treatment polypeptide.

31. nucleic acid construct, it includes according to claim 1 to the coded sequence of chimeric polyeptides described in any one of 30, institute It states coded sequence and is effectively connect with the regulating element that can be operated in host cell.

32. host cell contains nucleic acid construct according to claim 31.

33. host cell according to claim 32, wherein the host cell is prokaryotic host cell.

34. host cell according to claim 33, wherein the host cell is eukaryotic host cell.

35. the method for generating chimeric polyeptides complex, the method comprise the steps that in the item for being suitable for the formation of chimeric polyeptides complex Under part (for example, in aqueous solution) thus combination generates chimeric according to claim 1 to chimeric polyeptides described in any one of 30 Complex of polypeptides, the chimeric polyeptides complex include three chimeric polyeptides subunits and are characterized in that through each of chimeric polyeptides The double helix beam oligomerization that self-structure forms part is formed by Six helix bundle.

36. chimeric polyeptides complex, it includes according to claim 1 to three chimeric polyeptides subunits described in any one of 30 simultaneously And it is characterized in that the double helix beam oligomerization of the respective structure forming part point by chimeric polyeptides is formed by Six helix bundle.

37. complex according to claim 36, wherein each chimeric polyeptides subunit includes that enveloped virus merges born of the same parents Extracellular portion polypeptide, and wherein the complex includes purpose enveloped virus fusion protein (for example, wild type enveloped virus melts Hop protein) or enveloped virus fusion protein complex at least one merge before epitope, epitope is not present in wrapping before the fusion After the fusion of film virus amalgamation protein or fusion protein complex in form.

38. composition, it includes according to claim 1 to chimeric polyeptides described in any one of 30 or according to claim 36 or Chimeric polyeptides complex and pharmaceutical acceptable carrier, diluent or adjuvant described in claim 37.

39. identifying the substance (for example, small molecule or macromolecular) in conjunction with enveloped virus fusion protein or fusion protein complex Method, the method comprise the steps that make candidate substances with as above and the elsewhere this paper it is broadly described containing extracellular domain The chimeric polyeptides or chimeric polyeptides complex of polypeptide contact, wherein the extracellular domain polypeptide, which corresponds to enveloped virus, merges egg It is white, and detect the combination of candidate substances and chimeric polyeptides or chimeric polyeptides complex.

40. further including according to the method for claim 39, connecing candidate substances with fusion protein or fusion protein complex Touch and detect the combination of candidate substances and fusion protein or fusion protein complex.

41. according to method described in claim 39 or claim 40, wherein the candidate substances are library of compounds (examples Such as, small molecule or macromolecular library) part.

42. the method according to any one of claim 39 to 41 further includes separating candidate substances from library.

43. the method according to any one of claim 39 to 42, wherein the candidate substances and chimeric polyeptides or chimeric Complex of polypeptides specific binding.

44. the method according to any one of claim 39 to 43, wherein the candidate substances and fusion protein or merging Protein complexes specific binding.

45. generate with the antigen binding molecules of the immune interaction of enveloped virus fusion protein or fusion protein complex (for example, Antibody, such as neutralizing antibody) method, the method comprise the steps that (1) is with according to claim 1 to described in any one of 28 Chimeric polyeptides containing extracellular domain polypeptide contain extracellular domain according to claim 36 or claim 37 The chimeric polyeptides complex of polypeptide or its composition immunity inoculation animal according to claim 38, wherein extracellular structure Domain polypeptide corresponds to enveloped virus fusion protein；(2) from animal identification and/or separation and fusion protein or fusion protein complex The B cell of immune interaction；(3) antigen binding molecules expressed by this B cell are generated.

46. the antigen binding molecules generated by claim 45 the method, or with the antigen binding molecules with identical Epitope binding specificity derivative antigen binding molecules.

47. derivative antigen binding molecules according to claim 46, selected from antibody fragment (such as Fab, Fab ', F (ab’)₂, Fv), single-chain antibody (scFv) and domain antibodies (including such as shark antibody and camel class antibody), and comprising anti- The fusion protein of body, and any other improved configuration comprising antigen binding/recognition site immunoglobulin molecules.

48. immune regulation composite, it includes antigen binding molecules according to claim 46 or claim 47 and can Pharmaceutical carrier, diluent or adjuvant.

49. the method that excitation is directed to the immune response of enveloped virus fusion protein or fusion protein complex in subject, Described in method include merging extracellular structure containing envelope virus to subject's application is according to any preceding claims The chimeric polyeptides complex or composition in domain, wherein the extracellular domain polypeptide moiety of the chimeric polyeptides complex corresponds to packet Film virus amalgamation protein.

50. the method that excitation is directed to the immune response of enveloped virus fusion protein or fusion protein complex in subject, Described in method include to subject application can express it is according to any preceding claims containing envelope virus merge The chimeric polyeptides complex of extracellular domain or the DNA vaccination of composition or viral vectors/replicon, wherein the chimeric polyeptides The extracellular domain polypeptide moiety of complex corresponds to enveloped virus fusion protein.

51. the method for treating or preventing infection of coated virus in subject, wherein the method includes effective to subject's application The chimeric polyeptides complex of the fusion extracellular domain according to any preceding claims containing envelope virus of amount, and/ Or the antigen binding molecules according to claim 46 or claim 47 and/or combination according to claim 48 Object.

52. chimeric polyeptides, it includes downstreams to stabilize the protein molecule that part is effectively connect with heterologous structural, described heterologous Property structure stabilization part include complementary the first heptapeptide repetitive sequence area (HR1) and second area heptapeptide repetitive sequence (HR2), institute Complementary the first heptapeptide repetitive sequence area (HR1) and the second heptapeptide repetitive sequence area (HR2) are stated in the condition for being suitable for their associations Under (for example, in aqueous solution) associated each other to form antiparallel double helix beam.

53. the method for generating chimeric polyeptides complex, the method comprise the steps that in the item for being suitable for the formation of chimeric polyeptides complex Combination chimeric polyeptides according to claim 52 under part (for example, in aqueous solution), thus generate chimeric polyeptides complex, The chimeric polyeptides complex includes three chimeric polyeptides subunits and is characterized in that being formed by each self-structure of chimeric polyeptides Partial double helix beam oligomerization is formed by Six helix bundle.

54. chimeric polyeptides complex it includes three chimeric polyeptides subunits according to claim 52 and is characterized in that Six helix bundle is formed by by the double helix beam oligomerization of the respective structure forming part point of chimeric polyeptides.

Technical field

This application claims the Australian provisional applications of entitled " chimeric molecule and application thereof " that on March 30th, 2017 submits Numbers 2017901152 priority, the content of the document are completely incorporated herein by reference by reference.

This invention relates generally to the chimeric polyeptides based on virus membrane-fusion protein.More particularly it relates to comprising The virion surface expose portion and heterologous structural of virus amalgamation protein stabilize that the chimeric polyeptides of part, to be related to these chimeric The complex of polypeptide.It is merged the invention further relates to these complexs purposes in the composition and for exciting for enveloped virus The immune response of albumen or fusion protein complex and/or method for treating or preventing infection of coated virus.The present invention into One step is related to heterologous structural and stabilizes the purposes for partially making heterologous molecules of interest oligomerization.

Background of invention

Enveloped virus needs disease such as human immunodeficiency virus (HIV), influenza virus and respiratory syncytial virus (RSV) (RSV) Malicious film is merged with the film of host cell to enter and infect host cell.By occurring from meta-stable ' before fusion ' conformation to height The advantageous structural rearrangement of energy of stability ' after fusion ' conformation, virus amalgamation protein promote this process.This structural change The fusion for changing driving virus and host cell membrane, causes releasing virus genome to enter in cell.

Virus amalgamation protein is divided into currently based on the characterization of molecules of its respective structural framework and driving fusion process Three classifications.I class and Group III fusion protein are that conformation is tripolymer before its fusion and after fusion, and II class fusion protein Before fusion in conformation be dimer, conformation is then rearranged into form after trimeric fusion before the fusion.However, it is likely that The new category virus amalgamation protein for sharing some key features identical as the classification that these are defined at present can be identified in future.I Class and Group III fusion protein share basic Structural Characteristics, including N-terminal signal sequence and C-terminal cross-film and cytoplasmic domains.It Also share similar syncretizing mechanism, wherein before initial fusion tripolymer generating unit decompose from allow for formed fusion after three Obvious structural rearrangement required for aggressiveness.

Virus amalgamation protein is excellent subunit vaccine candidate, because they are many medically important coating diseases The main target of the protectiveness neutralizing antibody reaction of poison.However, the intrinsic meta-stable property of fusion protein, is design effectively subunit The major obstacle of vaccine, because evidence is it has been shown that in the extensive cross reactivity and strength excited during natural infection recently With property antibody mainly with merge before form reaction and not with merge after form react.In addition, viral envelope fusion protein melts Form contains before closing be not present in fusion after formal epitope (for example, Magro et al., 2012. Proc.Natl.Acad.Sci.USA 109(8):3089-3094).It is, therefore, usually considered that the stabilized fusion for vaccine Preceding form more caters to the need in antigenicity.But the traditional scheme for recombinantly expressing these protein generally results in too early triggering With conformation transition at form after the more stable fusion of structure.

Therefore, urgent need generates the new departure for stabilizing recombination fusion protein, and the recombination fusion protein is substantially Form is before still keeping it to merge to stimulate the immune response for being more effectively directed to enveloped virus.

Summary of the invention

The present invention be originated from it is identified below: can by fusion protein virion surface exposure structural domain (also known as Make " fusion extracellular domain polypeptide " or " extracellular domain ") downstream effectively connects heterologous moiety and carrys out simulated virus fusion protein Conformation before merging, the heterologous moiety include that a pair is associated each other to form the complementary heptapeptide of antiparallel double helix beam and repeat sequence Arrange area.A kind of ' molecule clamp ' is served as in this part, makes after merging extracellular domain stabilizing polypeptides and it being inhibited to be rearranged into fusion Conformation.Molecular Tweezers clamping method of the invention has been used to generate simulates influenza, RSV, HIV, measles virus and ebola disease respectively The chimeric polyeptides of conformation before the fusion of poison, and be consequently adapted to as the viral envelope fusion protein for generating the conformation before merging The quasi- platform like object.The chimeric polyeptides so generated can with self-assembly to form artificial enveloped virus fusion protein complex, The complex includes the oligomer of chimeric polyeptides and simulates structure before the fusion of enveloped virus native fusion proteins complex As.This self-assembly allows to generate artificial complex easily, especially in recombinant expression system so.It is chimeric more using the present invention Peptide generate artificial complex can be used for stimulate for enveloped virus native fusion proteins complex immune response (including formed Neutralizing antibody) method and composition in, as be described hereinafter.

Therefore, in one aspect, the present invention provides a kind of chimeric polyeptides, and it includes downstreams and heterologous structural stabilisation portion Divide the enveloped virus fusion extracellular domain polypeptide effectively connected, the heterologous structural stabilizes first of part comprising complementation The heptapeptide repetitive sequence area (HR1) and second area heptapeptide repetitive sequence (HR2), the first heptapeptide repetitive sequence (HR1) of the complementation Area and the second heptapeptide repetitive sequence area (HR2) be suitable for they associate under conditions of (for example, in aqueous solution) associate each other with Form antiparallel double helix beam.The general lack of complementarity with extracellular domain polypeptide in the area HR1 and the area HR2, thus their preferences Ground is formed, each other without forming antiparallel double helix beam with the structural member of extracellular domain polypeptide.In some embodiments In, each independently characterized by the n times of amino acid classes repeat 7 residue patterns, the pattern is expressed as the area HR1 and the area HR2 (a-b-c-d-e-f-g-)_nOr (d-e-f-g-a-b-c-)_n, wherein pattern element ' a ' to ' g ' refers to that it is in amino acid classes Conventional septuple position and n are equal to or greater than 2 number, and at least the 50% of conventional septuple position ' a ' and ' d ' is (or extremely Few 51% whole whole number percentages at least 99% and between them) it is occupied by hydrophobic amino acid type and conventional septuple At least 50% (or at least 51% at least 99% and whole integers hundred between them of position ' b ', ' c ', ' e ', ' f ' and ' g ' Score) it is occupied by hydrophilic amino acid type, point between resulting hydrophobic amino acid type and hydrophilic amino acid type Cloth makes it possible to identify heptapeptide repetitive sequence area.In some embodiments, one or both includes interior in the area HR1 and the area HR2 Source property I class enveloped virus fusion protein heptapeptide repetitive sequence region amino acid sequence is made from it or consisting essentially of.At this In the representative example of a type, the area HR1 and the area HR2 separately include the complementation of one or more I class enveloped virus fusion proteins The endogenous area (HRA) heptapeptide repetitive sequence A and the area heptapeptide repetitive sequence B (HRB), be made from it or consisting essentially of. In some embodiments, HRA region amino acid sequence and HRB region amino acid sequence are merged derived from identical I class enveloped virus Albumen.In other embodiments, HRA region amino acid sequence and HRB region amino acid sequence are derived from different I class enveloped virus Fusion protein.In representative example, the area HR1 and the area HR2 are independently selected from orthomyxovirus, paramyxovirus, retrovirus, hat The area HRA and the area HRB of the fusion protein of shape virus, filamentous form virus and Arenavirus expression.

In some embodiments, extracellular domain polypeptide corresponds to I class enveloped virus fusion protein extracellular domain.In In the representative example of this type, extracellular domain polypeptide includes one or two in the area endogenous HRA and the area endogenous HRB Person.Unrestricted enveloped virus with I class fusion protein include orthomyxovirus, paramyxovirus, retrovirus, coronavirus, Filamentous form virus and Arenavirus.

In other embodiments, extracellular domain polypeptide corresponds to Group III enveloped virus fusion protein extracellular domain. Representative enveloped virus with Group III fusion protein includes rhabdovirus and herpesviral.

Extracellular domain polypeptide (for example, I class or Group III) may include intact precursor extracellular domain polypeptide or part thereof Or it is made from it.In some embodiments, extracellular domain polypeptide lacks the endogenous of endogenous signal peptides, extracellular domain Head point, the endogenous stem portion of extracellular domain, endogenous mucin spline structure domain, endogenous film proximal end perimeter and interior Any one in source property fusogenic peptide or more persons.Extracellular domain polypeptide suitably includes epitope before at least one is merged, the fusion Preceding epitope is not present in after the fusion of enveloped virus fusion protein corresponding with extracellular domain polypeptide in form.

In some embodiments, the area HR1 of structure stabilization part is connected with the area HR2 by connector, and the connector is usual It is made of about 1 to about 100 amino acid residue (and whole integer amino acid residues between them), and generally by about 1 It is formed to about 100 amino acid residues (and whole integer amino acid residues between them).Connector may include at least one A such part, the part, which is selected from, promotes the purification part of chimeric polyeptides purifying, metering needle to answer the immune of chimeric polyeptides The cell-targeting part of the immunological regulation part, guide chimeric polyeptides to specific cells hypotype answered and the portion for assigning configuration flexibility Point.

Chimeric polyeptides can be generated synthetically or by recombinant means.In the embodiment that recombination generates chimeric polyeptides, The present invention provides nucleic acid construct on the other hand, and it includes volumes such as above and the broadly described chimeric polyeptides in the elsewhere this paper Code sequence, the coded sequence are effectively connect with the regulating element that can be operated in host cell.

In a related aspect, the present invention provides a kind of host cell, contains above and the elsewhere this paper is broadly described Nucleic acid construct.Host cell can be protokaryon or eukaryotic host cell.

Heterologous structural stabilizes part in addition to after stablizing the confrontation of extracellular domain polypeptide and being rearranged into fusion in terms of conformation Practicability except, heterologous structural, which stabilizes part, can also be used to making any heterologous molecules of interest oligomerization chemical conversion oligomer, especially Its tripolymer.Therefore, on the other hand, the present invention provides a kind of such as above and broadly described chimeric polyeptides in the elsewhere this paper, It includes downstreams to stabilize the protein molecule that part is effectively connect with heterologous structural, and the heterologous structural stabilizes part Include complementary the first heptapeptide repetitive sequence area (HR1) and second area heptapeptide repetitive sequence (HR2), the first heptapeptide of the complementation The repetitive sequence area (HR1) and the second heptapeptide repetitive sequence area (HR2) are under conditions of being suitable for them and associating (for example, in aqueous solution In) associated each other to form antiparallel double helix beam.

Chimeric polyeptides of the invention can under suitable conditions (for example, in aqueous solution) self-assembly to form chimeric polyeptides Complex.Therefore, on the other hand, the present invention provides a kind of method for generating chimeric polyeptides complex, wherein the method It include: that combination is such as above under conditions of (for example, in aqueous solution) being suitable for the formation of chimeric polyeptides complex and the elsewhere this paper is wide The chimeric polyeptides that justice defines, thus generate chimeric polyeptides complex, the chimeric polyeptides complex include three chimeric polyeptides simultaneously And it is characterized in that the double helix beam oligomerization of the respective structure stabilization part by chimeric polyeptides is formed by Six helix bundle.

In a related aspect, the present invention provides a kind of chimeric polyeptides complex, and it includes such as above and the elsewhere this paper is wide Three chimeric polyeptides of general description and it is characterized in that few by the double helix beam of the respective structure stabilization part of chimeric polyeptides Dimerization is formed by Six helix bundle.In some embodiments that chimeric polyeptides include enveloped virus fusion extracellular domain polypeptide In, chimeric polyeptides complex is formed by the chimeric polyeptides self-assembly, the chimeric polyeptides complex includes purpose coating disease Epitope before at least one of malicious fusion protein (for example, wild type enveloped virus fusion protein) or fusion protein complex merge , epitope is not present in after the fusion of enveloped virus fusion protein or fusion protein complex in form before the fusion.

In another related fields, the present invention provides a kind of composition, and it includes such as describe extensively with the elsewhere this paper above Chimeric polyeptides or chimeric polyeptides complex and pharmaceutical acceptable carrier, diluent or adjuvant.In some embodiments, composition is Immune regulation composite.

In some embodiments that chimeric polyeptides include enveloped virus fusion extracellular domain polypeptide, of the invention is chimeric Complex of polypeptides can be used for the excitation in subject or production animal and be directed to enveloped virus fusion protein or fusion protein complex Immune response.Therefore, another aspect of the present invention provides one kind excitation in subject and is directed to enveloped virus fusion protein Or the method for the immune response of fusion protein complex, wherein this method includes wide to subject's application such as above and elsewhere this paper The chimeric polyeptides complex or composition of general description, wherein the extracellular domain polypeptide of chimeric polyeptides complex corresponds to coating disease Malicious fusion protein.

In a related aspect, the present invention provides a kind of identify and enveloped virus fusion protein or fusion protein complex knot The method of the substance (for example, small molecule or macromolecular) of conjunction, the method comprise the steps that making candidate substances and such as above and this paper The broadly described chimeric polyeptides containing extracellular domain polypeptide in elsewhere or chimeric polyeptides complex contact, wherein extracellular domain Polypeptide corresponds to enveloped virus fusion protein, and the combination of detection candidate substances and chimeric polyeptides or chimeric polyeptides complex.In In specific embodiment, this method further includes contacting candidate substances with fusion protein or fusion protein complex and detecting time Select the combination of substance and fusion protein or complex.In a particular embodiment, candidate substances are library of compounds (for example, small Molecule or macromolecular library) part.In some embodiments of these embodiments, this method further includes separating from library Candidate substances.Suitably, candidate substances and chimeric polyeptides or chimeric polyeptides complex and preferably fusion protein or fusion protein Complex specific binding.

In another related fields, the present invention provides a kind of generate and enveloped virus fusion protein or fusion protein complex The method of the antigen binding molecules (for example, antibody such as neutralizing antibody) of immune interaction, the method comprise the steps that (1) is used Such as chimeric polyeptides or chimeric polyeptides complex above and the broadly described polypeptide containing extracellular domain in the elsewhere this paper or combination Object immunity inoculation animal, wherein extracellular domain polypeptide corresponds to enveloped virus fusion protein；(2) from animal identification and/or point From the B cell with fusion protein or the immune interaction of fusion protein complex；(3) it generates and is resisted by what this B cell was expressed Former binding molecule.

A kind of antigen binding molecules are also provided as an additional aspect of the present invention, it is extensive by the above and elsewhere this paper The methods of vaccination of description generates or derivative antigen binding molecules, has epitope knot identical with antigen binding molecules Close specificity.Derivative antigen binding molecules can selected from antibody fragment (such as Fab, Fab ', F (ab ')₂, Fv), single-stranded (scFv) With domain antibodies (e.g., including shark antibody and camel class antibody (camelid antibodies)), and include antibody Fusion protein, and any other improved configuration comprising antigen binding/recognition site immunoglobulin molecules.

In a related aspect, the present invention provides a kind of immune regulation composite, and it includes such as above and the elsewhere this paper is wide Antigen binding molecules and pharmaceutical acceptable carrier, the diluent or adjuvant of general description.

Theme such as the broadly described fusion extracellular domain polypeptide containing envelope virus in the above and elsewhere this paper is chimeric more Peptide or its complex and composition and antigen binding molecules can also be used to treat or prevent infection of coated virus.Therefore, again In one aspect, the present invention provides a kind of method that infection of coated virus is treated or prevented in subject, wherein the method Method includes merging extracellular knot containing envelope virus as the above and elsewhere this paper is broadly described to subject's application is a effective amount of The chimeric polyeptides of structure domain polypeptide or its complex, composition or antigen binding molecules.

Brief description

Fig. 1 is the reactive diagram of ELISA RSV F shown with conformation specific monoclonal antibody.A. special before fusion Property monoclonal antibody D25 (Zhao et al., Proc.Natl.Acad.Sci.USA 2000.97 (26): 14172-7) with 23.4 ± The affinity of 12.5nM does not stabilize F extracellular domain F with corresponding in conjunction with the stabilized chimeric RSV F of clamp, but not Sol is combined.B. merge before monoclonal antibody specific D25 with comparativity affinity (be respectively 1.2 ± 0.2nM and 1.3 ± 0.2nM) it is fitted into conjunction with RSV F with stabilized compare of the stabilized chimeric RSV F DS cav mutant of clamp and folding, but It is not stabilized in conjunction with F extracellular domain F sol with corresponding.C. specific monoclonal is anti-before extensive cross-neutralization, fusion Body MPE8 (Corti et al., Nature 2013.501 (7467): 439-43) (is 7.6 ± 1.5nM respectively with comparativity affinity RSV F is fitted into stabilized compare of the stabilized chimeric RSV F DS cav mutant of clamp and folding with 10.8 ± 2.4nM) In conjunction with, but do not stabilized in conjunction with F extracellular domain F sol with corresponding.

Fig. 2 is the reactive diagram of ELISA influenza H3 shown with conformation specific monoclonal antibody.A. head specificity Antibody C05 (Ekiert et al., Nature 2012.489 (7417): 526-32) is similarly tied with H3sol, H3 clamp and QIV) It closes, however stem monoclonal antibody specific CR8043 (Friesen et al., Proc.Natl.Acad.Sci.USA before merging 2014.111 (1): 445-50) with the affinity of 3.3 ± 0.8nM in conjunction with the stabilized Chimeric influenza HA of clamp, but not with phase The HA extracellular domain or business QIV answered combine.

Fig. 3 is the diagram for showing the size exclusion chromatography separation of protein oligomeric state.The stabilized Chimeric influenza of clamp HA exists as soluble tripolymer, is such as washed in about 11mL from Superdex 200Increase 10/300GL column by it Seen in de-.In contrast, the corresponding HA extracellular domain expressed when separation at about 8mL and 12mL from the column elute, this with A part of protein exists as aggregation and another part is consistent as monomer presence.Previously it has been shown that influenza Form can be dissociated into monomeric form or aggregation (Weldon et al., PLoS One by the exposure of fusogenic peptide after the fusion of HA 2010.5(9)).The business QIV prompt high molecular weight eluted at 8mL assembles object.

Fig. 4 is the diagram of the neutrality immune response generated when showing inoculation.Mouse from business (QIV) vaccine inoculation Serum show with about 180 IC₅₀Value neutralizes Flu-A/Baoding Anguo/51/2010 (H3N2).In contrast, it clamps It presss from both sides stabilized Chimeric influenza HA and shows that viral neutralization activity level greatly increases, IC₅₀Value be 1:14,000 (95%CI 11, 000-17,000), and the serum of the mouse from the inoculation of corresponding HA extracellular domain even the maximum dose level 1:20 of test not Show neutralization activity.H3N2 is not influenced with H3sol, QIV precincubation to neutralize, however is eliminated H3N2 with H3 clamp precincubation and neutralized.

Fig. 5 is the diagram of the subdomain specificity for the immune response for showing induction.From H3sol, H3 clamp or business The serum of the mouse of vaccine (QIV) inoculation is shown and the difference reaction of the head subdomain of H3 and stem's subdomain.Come From the serum of the mouse of the stabilized Chimeric influenza HA immunity inoculation of clamp show for stem's structural domain maximum reactivity (about 25% total H3 specificity response), and for the serum of the mouse from H3sol and QIV immunity inoculation, this percentage is low to be obtained More (being 1% and 4% respectively).

Fig. 6 is the diagram for showing H5 cross reactivity.ELISA is used to measure the reactivity with H5.End point titres are calculated as It generates and is greater than the reactive greatest dilution of+3 standard deviations of background.From H3sol, H3 clamp, commercial vaccine (QIV), H1 pincers The serum of the mouse of folder and the inoculation of H5 clamp is shown and the difference reaction of H5.It is small from H3 clamp or H1 clamp immunity inoculation The serum of mouse is shown and the cross reactivity of H5 ratio QIV much bigger (being 27 times of growths and 81 times of growths respectively).

Fig. 7 is the diagram for showing the subdomain of responsible H5 cross reactivity.ELISA is used to measure the reactivity with H5. End point titres, which are calculated as generating, is greater than the reactive greatest dilution of+3 standard deviations of background.It shows anti-to total H5 of mice serum Answering property (grey column).Stem's specific antibody (white column) is adsorbed in advance before ELISA using the precincubation of serum and H3 stem Or addition monoclonal antibody FI6v3 wins stem's specific antibody (black column) so that competing.

Fig. 8 is the diagram for showing the stabilized soluble tripolymer MERS spike protein purifying of clamp.A. from CHO after expressing The clamp of supernatant purifying stabilizes the SDS-PAGE of MERS furcella.It is in the protein band of about 200kDA relative to institute Obtaining monomer MERS albumen (glycan comprising engagement) is correct approx. dimension, and in the control purifying from CHO supernatant In the invisible albumen.B. clamp stabilizes size exclusion of the MERS spike protein on 6 10/300GL column of Superdex Chromatography.Main protein elutes display relative to non-agglomerated, tripolymer MERS albumen (glycan comprising engagement) at 12.5mL For the about 600kDA of correct size.

Fig. 9 is to show that the clamp for lacking mucin spline structure domain from the purifying of CHO supernatant stabilizes Ebola GP albumen Diagram.The SDS-PAGE of no DTT is analyzed relative to resulting Ebola GP albumen (GP1 is connected with GP2 by natural disulphide bonds) (glycan comprising engagement) is that the about 100kDA of correct size shows a protein band.There is the SDS-PAGE of DTT to analyze The Ebola GP albumen (glycan comprising engagement) sheared relative to furin be correct size about 60kDA and 30kDa shows two protein bands.

Figure 10 is to show (to lack mucin spline structure with the ELISA Ebola GP clamp of height neutralizing monoclonal antibody Domain) reactive diagram.Monoclonal antibody Kz52,1H3,2G4,4G7 and 13C6 (Murin et al., PNAS.2014 11 (48): 17182-7) combined with high-affinity and Ebola GP clamp (lacking mucin spline structure domain).

Figure 11 is the diagram for showing the thermal stability of Ebola GP clamp (lacking mucin spline structure domain).The Ai Bo of purifying Draw GP clamp (lacking mucin spline structure domain) dry in conjunction with elisa plate and in the presence of 30% sucrose.Directly or 37 DEG C incubate 14 days after measurement with height neutralizing monoclonal antibody Kz52,4G7 and 13C6 (Murin et al., PNAS.2014 11 (48): 17182-7 reactivity).Reactivity is observed without significant change, this shows that the stabilized protein of clamp is steady in high temperature Surely continue the extended time.

Figure 12 was shown in after BALB/C mice immunity inoculation for Ebola GP clamp (lacking mucin spline structure domain) Immune response diagram.By 3 groups, every group of 5 mouse 1 μ g Ebola GP clamp (lacking mucin spline structure domain), 1 μ g Ebola GP clamp (lacking mucin spline structure domain)+3ug saponin adjuvant Quil A or the PBS intradermal immunization as negative control Inoculation.In the 0th, the 28th and the 56th day immunized mice 3 times and at the 27th day (bloodletting 1), the 55th day (bloodletting 2) and the 84th Its (bloodletting 3) acquires blood.A. it is quantified by ELISA special to Ebola GP clamp (lacking mucin spline structure domain) in serum Antibody.B. the final dilution that the reading higher than+3 standard deviations of background can be generated by calculating, calculates end point titres.

Figure 13 was shown in after BALB/C mice immunity inoculation for Ebola GP clamp (lacking mucin spline structure domain) Neutralize the diagram of the immune response ability of zaire strain of Ebola virus living.(lack mucin sample from 1 μ g Ebola GP clamp Structural domain) serum of mouse of+3 μ g saponin adjuvant Quil A immunity inoculations can neutralize Ebola virus living.Lead to plaque shape Being computed at the geometric mean titer that unit reduces by 50% is 52.8 (95%CI 24.5-114.0).

Figure 14 is to show the diagram for making jaw arrangement domain that silencing be immunized by being incorporated to N- linked glycosylation site.In Ebola GP In clamp (lacking mucin spline structure domain), four independences are generated inside the HRB of the clamp sequence based on HIV GP160 base SSM Mutation.It tests for Ebola GP clamp (lacking mucin spline structure domain) and is immunized with from the stabilized Chimeric influenza HA of clamp The serum reactivity of the mouse of inoculation, the identical clamp sequence of the Ebola GP clamp or four potential sites together Enter glycosylated clamp sequence stabilization.Reactivity is glycosylated at every kind of independent site and is significantly reduced, to support following Assuming that: this method can be used to reduce the reactivity to jaw arrangement domain.Mutation is incorporated to by measurement Kz52 affinity confirmation The Ebola GP (lacking mucin spline structure domain) of clamp sequence is correctly folded.

Figure 15 is to show that the clamp from eight kinds of viral SDS-PAGE purifying stabilizes the photograph displaying of antigen.By orange The band that arrow is directed toward indicates the product that do not shear and yellow arrows indicate the product of shearing.The estimated molecular weight of antigen is: Influenza HA clamp=about 85kDa, RSV F clamp=about 65kDa, Ebola GP clamp=about 72kDa, clamp=about 64 Buddhist nun pa F, MERS S clamp=about 200kDA, drawing sand GPC clamp=about 75kDA, morbilli=about 65kDa and HSV2-Gb clamp=about 100kDa。

Figure 16 is the diagram of the result of mouse Protective strategy after showing influenza virus H1N1pdm attack: (A/B) mouse (n= 5) it is folded with H1sol, H1 derived from strain Cal/09 (H1N1pdm) or H1 clamp is inoculated with and then with matched influenza poison Strain attack.(C/D) mouse use derived from strain Switz/13 (H3N2) H3sol, H3 fold or H3 clamp inoculation and then It is attacked with divergence strain Cal/09 (H1N1pdm).

Figure 17 is shown in the diagram that 43 DEG C of clamps for continuing 72 hours stabilize the thermal stability of antigen.Clamp is stablized The vaccine candidate object and reference protein of change incubate 72 hours at 43 DEG C, and using with three sufficiently characterize for every kind of antigen MAb measurement of the reactivity as thermal stability.A. compare RSV F using specificity mAb D25, AM22 and MPE8 before merging Clamp and using fold trimerising domain and it is structure-based stabilize mutation alternative (McLellan et al., Science, 2013.342 (6158): the 592-8 pages).The head B.HA stem specificity mAb FI6V3 and CR6261 and HA is special Property mAb 5J8 be used to compare influenza H1 clamp and using fold trimerising domain (foldon trimerization Domain) alternative.

Figure 18 is the diagram for showing the immune response of the stabilized subunit vaccine of clamp.Test from RSV F clamp, Fsol or Fdscav folds serum (McLellan et al., Science, 2013.342 (6158): the 592- of the mouse of immunity inoculation 598) ability of RSV is neutralized.

Figure 19 be show be included in molecule clamp promote trimeric fusion before conformational stability figure and photograph show.Pass through size The stabilized Nipah virus fusion glycoprotein of exclusion chromatographic analysis clamp.Elution volume and solubility on 200 column of Superdex The expection molecular weight of trimer protein is related.There are albumen textures before uniform fusion for negative staining electron microscopy (illustration) confirmation As.

Figure 20 is the diagram for showing the immune response that subunit vaccine is stabilized for clamp.Test comes from Buddhist nun's pa F clamp In the serum of the mouse of immunity inoculation and the ability of Nipah virus.In all small figures, the value of display is the several of individual mouse What average, error line indicate geometric standard deviation.

Figure 21 is to show that molecule clamp is incorporated to the stable figure and photograph of Trimeric structures for promoting Group III virus amalgamation protein Mutually show.The stabilized HSV2 fusion glycoprotein B (gB) of clamp is analyzed by size exclusion chromatography.Washing on 6 column of superose Lift-off is long-pending related to the soluble expection molecular weight of trimer protein.There are uniform three for negative staining electron microscopy (illustration) confirmation Aggressiveness conformation, similar to the structure (Heldwein et al., Science, 2006.313 (5784): 217-20) delivered. Show that this conformation combines most of neutralizing antibodies (Cairns et al., JVi, 2014.88 (5): 2677-2689).

Detailed description of the invention

1. definition

Unless specified otherwise herein, whole term and scientific term used herein have with it is of the art those The normally understood equivalent of technical staff.Although the similar or equivalent any means to those described herein method and material It can be used for implementing with material or test the present invention, however describe preferred method and material.For the purposes, hereafter Define following term.

Article " one (a) " and " a kind of (an) " be used to refer to herein the article one or more than one (that is, at least One) grammar object.For example, " element " means an element or more than one element.

As used herein, "and/or" refers to and including any of one or more relevant institute's lists and all may group Close, and when by it is alternative (or) interpret when, refer to and including lack combine.

In addition, when mentioning mensurable value such as amount, dosage, time, temperature, activity, level, number, frequency, percentage, ruler Whens degree, size, amount, weight, position, length etc., what term " about " as used herein and " approximation " were intended to illustrate Amount, dosage, time, temperature, activity, level, number, frequency, percentage, scale, size, amount, weight, position, length etc. ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% variation.Wherein term " about " and " approximation " with In the case that the positioning in all regions or position are associated with use inside reference polypeptide, it is residual that these terms cover ± up to 20 amino acid Base, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 ammonia The variation of base acid residue, ± up to 3 amino acid residues, ± up to 2 amino acid residues or even ± 1 amino acid residue.

Term " adjuvant " as used herein refers to such compound, with immunogene specific in composition (for example, the present invention Chimeric polyeptides or complex) when being applied in combination, gained immune response will be promoted, including strengthen or widen antibody mediated immunity response With the specificity of one or both of cellullar immunologic response.

Term " substance (agent) " and " compound " herein exchange are used to refer to any compound or substance, such as but not It is limited to small molecule, nucleic acid, polypeptide, peptide, drug, ion etc.." substance " can be any chemicals, entity or part, including but It is not limited to synthesis and naturally occurring protein properties and non-proteinaceous entity.In some embodiments, substance is core Acid, nucleic acid analog, protein, antibody, peptide, aptamers, the oligomer of nucleic acid, amino acid or sugar, including but not limited to albumen Matter, oligonucleotides, ribozyme, deoxyribozyme (DNAzymes), glycoprotein, siRNA, lipoprotein, aptamers and its modifier and group Close etc..In some embodiments, nucleic acid is DNA or RNA and nucleic acid analog, for example, it may be PNA, pcPNA and LNA.Nucleic acid can be nucleic acid, oligonucleotides, PNA etc. single-stranded or double-stranded, and that can be selected from coding destination protein.These cores Acid sequence for example including but be not limited to coding and serve as the nucleic acid sequence of protein of transcription repression albumen, antisense molecule, ribozyme, small Inhibition nucleic acid sequence, such as, but not limited to, RNAi, shRNAi, siRNA, microRNA i (mRNA), antisense oligonucleotides etc..Egg White matter and/or peptide material or its segment can be any destination protein, such as, but not limited to, the protein of mutation；Therapeutic egg It is white；Truncated protein, wherein protein is not present in expressing in cell in cell or with reduced levels under normal circumstances.Mesh Albumen can include selected from the following group: the albumen of mutation, genetically engineered albumen, peptide, synthetic peptide, recombinant protein, chimeric egg White, antibody, humanized proteins' matter, humanized antibody, chimeric antibody, the protein of modification and its segment.

As used herein, refer to can be by specificity for term " antigen " and the equivalent statement (for example, " antigenicity ") of its grammer Compound, composition or the object of the product (such as antibody molecule or T cell receptor) of humoral immunity or cellular immunity specific binding Matter.Antigen can be any kind of molecule, for example including haptens, simple intermediate metabolites, sugar (for example, oligosaccharide), Lipid and hormone and macromolecular such as complicated sugared (for example, polysaccharide), phosphatide and protein.The antigen of common class includes but unlimited In viral antigen, bacterial antigen, fungal allergen, protozoon and other parasite antigens, tumour antigen, participates in itself and exempt from Antigen, toxin and other miscellaneous antigens of epidemic disease, allergy and transplant rejection.

" antigen binding molecules " mean the molecule for having binding affinity to target antigen.It should be appreciated that this term expansion To showing albumen texture derived from the immunoglobulin, immunoglobulin fragment and non-immunoglobulin of antigen-binding activity Frame.Can be used for implementing representative antigen binding molecules of the invention include polyclonal antibody and monoclonal antibody and its segment (such as Fab、Fab’、F(ab’)₂, Fv), single-stranded (scFv) and domain antibodies (e.g., including shark antibody and camel class antibody), and Fusion protein comprising antibody, and any other improved configuration comprising antigen binding/recognition site immunoglobulin molecules. Antibody includes the antibody of any classification, and such as IgG, IgA or IgM (or its subclass), and antibody needs not be any specific category.It takes Certainly in the antibody amino acids sequence of its heavy chain constant region, it is different classes of that immunoglobulin, which can incorporate into,.There are five major class Immunoglobulin: IgA, IgD, IgE, IgG and IgM, and can be further divided into subclass (of the same race by several in these classifications Type), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Heavy chain constant region corresponding to different classes of immunoglobulin It is referred to as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different classes of immunoglobulin are well known.Antigen binding Molecule also covers the antibody of homodimeric antibody and multivalent forms.In some embodiments, antigen binding molecules are inosculating antibodies Body, in the antibody part of heavy chain and/or light chain be originated from particular species or belong to the anti-of specific antibodies classification or subclass Corresponding sequence in body is identical or homologous, at the same the rest part of the chain be originated from another species or belong to another antibody Corresponding sequence in the segment of the antibody and this antibody of classification or subclass is identical or homologous, as long as they show desired life Object activity can be (see, e.g. U.S. Patent number 4,816,567；With Morrison et al., 1984, Proc.Natl.Acad.Sci.USA 81:6851-6855).It is contemplated that generally being determined by shifting complementation to people's variable domains Determine area (CDR) variable heavy chain and variable light from non-human (for example, rodent, preferably mouse) immunoglobulin to generate Humanized antibody.Common human antibody residue is then replaced in the framework region of non-human counterpart.Using derived from source of people The antibody component for changing antibody avoids potential problems relevant to the immunogenicity of non-human constant region.Clone inhuman immune globulin White variable domains, the general technology of especially rat immune globulin variable domains for example by Orlandi et al. (1989, Proc.Natl.Acad.Sci.USA 86:3833) description.The technology of Humanized monoclonal antibodies is generated for example by Jones et al. (1986, Nature 321:522), Carter et al. (1992, Proc.Natl.Acad.Sci.USA 89:4285), Sandhu (1992, Crit.Rev.Biotech.12:437), Singer et al. (1993, J.Immun.150:2844), Sudhir (are compiled Write), Antibody Engineering Protocols, Humana Press, Inc.1995), Kelley (" Engineering Therapeutic Antibodies, " quoted from Protein Engineering:Principles and Practice Cleland et al. (writes), the 399-434 pages (John Wiley&Sons, Inc.1996) and Queen et al. U.S. Patent number 5,693,762 (1997) description.Humanized antibody includes " primatized " antibody, and wherein the antigen binding domain of antibody is derivative personal Antibody caused by purpose antigen immunity inoculation macaque.Also design humanized antibody is as antigen binding molecules.

As used herein, term " antiparallel " refers to that wherein polymer areas or section are in parallel-oriented but have opposite pole The protein properties polymer of property.

As used herein, term " specific binding " refers at molecule (including macromolecular such as protein and other biological product) Inhomogenous group exist and make decision association reaction existing for chimeric polyeptides or complex of the present invention.In a particular embodiment, When referring to antigen binding molecules, term " specific binding " is used interchangeably with term " specific immunity interaction ", is referred to It is anti-to there is the existing combination of make decision chimeric polyeptides or complex of the present invention in the inhomogenous group of protein and other biological product It answers.Under specified determination condition, molecule is with chimeric polyeptides of the invention or complex specific binding and not with apparent Amount combines other molecules (for example, protein or antigen) present in sample.It is a variety of to exempt from antigen binding molecules embodiment Epidemic disease measuring method pattern can be used to the antigen knot for selecting to interact with chimeric polyeptides of the invention or complex specific immunity Close molecule.For example, solid phase ELISA immunoassay is routinely used to select the Dan Ke with the immune interaction of protein specific Grand antibody.About the description for the immunoassay format and condition that may be used to determine specific immunoreactivity, referring to Harlow With Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York。

When mentioning molecule in use, term " chimeric " means such molecule, the molecule, which contains, to be derived from, be obtained from or divide From from or part based on two or more Different Origins or source.Therefore, when polypeptide includes two of separate sources or more Multiple amino acid sequences and include (1) in nature not together existing polypeptide sequence (that is, in amino acid sequence at least it One relative at least one of other amino acid sequences be it is heterologous), or (2) do not adjoin naturally amino acid sequence when, it is embedding It closes.

" coded sequence " means the final mRNA product for facilitating the polypeptide product of encoding gene or gene (for example, montage The mRNA product of gene afterwards) any nucleic acid sequence.The polypeptide of encoding gene is not produced on the contrary, term " non-coding sequence " refers to Object contributes or not to the contributive any nucleic acid sequence of final mRNA product of gene.

Term " coiled coil " or " coiled-coiled structure " are used to refer to structural in protein interchangeably herein Motif, two of them or more alpha-helix (most often 2-7 alpha-helix) wind (dimer together as the stock of rope It is most common type with tripolymer).Many coiled coil type protein participate in important biological function, such as adjust gene table It reaches, such as transcription factor.Coiled coil often but not always contains the repeating sample of hydrophobicity (h) and polarity (p) amino acid residue Formula, hpphppp or hppphpp, referred to as heptapeptide repetitive sequence (see below).Sequence with this repeat pattern is folded into α- Helix secondary structure is caused hydrophobic residue as the ` band ` that the spiral is mildly wound is surrounded in a manner of left hand and presented, and forms two Close structure.It is that wrapping is clipped in hydrophily opposite to each other that best mode itself is arranged two such spiral in water-filled environment Hydrophobic chain between amino acid.Therefore exactly burying hydrophobic surface just provides the heat power driving force of alpha-helix oligomerization.Volume Curling in bent helical interface is unusually close.Alpha-helix can be parallel or antiparallel, and it is super that left hand is usually taken Spiral form.Although unfavorable, also in nature with observe some right handed coiled coils in the protein of design.Term " coiled coil " or " coiled-coiled structure " is that those skilled in the art are obvious based on common knowledge.With reference in this side The specific document in face is related to the paper of coiled-coiled structure to look back, such as example, Cohen and Parry (1990.Proteins 7: 1-15)；Kohn and Hodges (1998.Trends Biotechnol 16:379-389)；Schneider et al. (1998.Fold Des 3:R29-R40)；Harbury et al. (1998.Science 282:1462-1467)；Mason and Arndt (2004.Chem-BioChem 5:170-176)；Lupas and Gruber (2005.Adv Protein Chem 70:37-78)； Woolfson(2005.Adv Protein Chem 70:79-112)；Parry et al. 2008.J Struct Biol 163: 258-269)；With Mcfarlane et al. (2009.Eur J Pharmacol 625:101-107).

As used herein, term " complementary " and its equivalent statement of grammer refer to hybridize each other, oligomerization (for example, Dimerization), interaction or otherwise form two or more structural members of complex (for example, peptide, polypeptide, nucleic acid, small Molecule or part thereof etc.) feature.For example, " polypeptide complementary region " can be formed together in a particular embodiment with The complex that antiparallel double helix beam is characterized.

As used herein, term " complex " refers to directly with one another and/or the molecule of mediate contact (for example, peptide, polypeptide etc.) Collection polymers or aggregation.In a particular embodiment, " contact " or more specifically, " directly contact " means two or more Molecule is close enough, thus attract noncovalent interaction, as Van der Waals for, hydrogen bond formed, ionic interaction and Hydrophobic interaction etc. dominates the interaction of molecule.In this kind of embodiment, molecule (for example, peptide and polypeptide) it is compound Body is formed under such conditions, thus complex it is thermodynamically advantageous (for example, with its modular molecular do not assemble or it is not multiple Conjunction state is compared).As used herein, unless otherwise term " complex " refers to two or more molecule (examples such as in addition description Such as, peptide, polypeptide or combinations thereof) collection polymers.In a particular embodiment, term " complex " refers to the collection polymers of three polypeptides.

Term " library of compounds " as used herein refers to that any set of compound, the set are different comprising structure Different kinds of molecules.Library of compounds may include combinatorial chemistry library or natural product libraries.It is (including non-covalent by interaction Interaction, for example, by hydrogen bond, ionic bond, Van der Waals attract or hydrophobic interaction), can with it is of the invention embedding Polypeptide or composite bulk phase interaction are closed, can reside in library of compounds in conjunction with or to its any type molecule with affinity In.For example, the library of compounds that the present invention covers can contain naturally occurring molecule, such as sugar, monosaccharide, oligosaccharide, polysaccharide, ammonia Base acid, peptide, oligopeptides, polypeptide, protein, receptor, nucleic acid, nucleosides, nucleotide, oligonucleotides, polynucleotides, including DNA and DNA Segment, RNA and RNA segment etc., lipid, vitamin A acid, steroids, glycopeptide, glycoprotein, proteoglycans etc.；Or naturally occurring molecule Analog or derivative (such as peptide mimics)；With non-naturally occurring molecule, such as generated using such as combinatorial chemistry technique " small molecule " organic compound；And its mixture.

Throughout the whole instruction, unless the context requires otherwise, otherwise word "comprising", " containing " and " including " will be managed Solution at showing to include the steps that described or element or the step or element group, but be not excluded for any other step or Element or any other step or element group.Therefore, indicate that listed element is to need or strong using term "comprising" etc. System, but other element is optional and may exist or can be not present." by ... form " mean to include and limit In phrase " by ... form " after any object.Therefore, phrase " by ... form " indicate listed element be need or It is compulsory, and other element cannot exist.By " substantially by ... form " mean include after the phrase it is listed Any element, and it is limited to other of the activity of concrete regulation or effect in the disclosure for not interfering or being helpless to listed elements Element.Therefore, phrase " substantially by ... form " indicates that listed element is to need or compulsory, but other element is to appoint Choosing and may exist or can be not present, this depends on whether they influence the activity or effect of listed elements.

As used herein, by no matter which kind of means (including chemically conjugated or recombinant means by heredity (for example, melted Close)) under the context that makes two more multicomponents or component or structural domain be bonded together, term " conjugation ", " connection ", " fusion " Or " fusion " and its grammatical equivalents are used interchangeably.(for example, using heterobifunctional agents) chemical conjugation methods are this fields It is known.More specifically, as used herein, " enveloped virus fusion protein extracellular domain "-" structure stabilization part " fusion Object or conjugate refer to appropriate enveloped virus fusion protein extracellular domain and stable structure in meta-stable, merging preceding conformation Change the heredity conjugation or chemically conjugated of part.In a particular embodiment, structure stabilization part is through connector (such as glycine-silk Propylhomoserin (gly-ser) connector) it is merged indirectly with enveloped virus fusion protein extracellular domain.In other embodiments, structure Part is stabilized directly to merge with enveloped virus fusion protein extracellular domain

" conservative amino acid displacement " is that wherein amino acid residue being set with what the amino acid residue with similar side chain was replaced It changes.The defined amino acid residue families with similar side chain in this field, these families usually can be broken down as follows:

1 amino acid subclassification of table

Conservative amino acid displacement further includes the grouping based on side chain.For example, the amino acid group with aliphatic lateral chain is sweet Propylhomoserin, alanine, valine, leucine and isoleucine；Amino acid group with aliphatic-hydroxyl side chains is serine and Soviet Union's ammonia Acid；Amino acid group with beta-branched side is asparagine and glutamine；Amino acid group with aromatic side chains is phenylpropyl alcohol Propylhomoserin, tyrosine and tryptophan；Amino acid group with basic side chain is lysine, arginine and histidine；With with sulfur-bearing The amino acid group of side chain is cysteine and methionine.For example, being reasonably desirable to bright with isoleucine or valine replacement Propylhomoserin is similarly replaced with glutamic acid replacement aspartic acid, with serine replacement threonine or with amino acid relevant in structure Amino acid will not generate significant impact to the performance of obtained variant polypeptide.Whether amino acid variation generates functional polypeptide It can be determined easily by the activity of analysis polypeptide.It is shown under the gauge outfit of " exemplary permutation and preferred displacement " in table 2 Preservative replacement.In general, the displacement of notable difference, completion do not belong to this hair in terms of by selecting its influence to following aspect Amino acid replacement in bright range: structure, (b) molecule charge target site at of (a) peptide backbone in replacement areas is maintained Or hydrophobicity or (c) side-chain bulk.After introducing displacement, the biological activity of variant is screened.

Table 2 illustratively and preferred amino acid replacement

Term " construct " refers to the genetic recombination point comprising one or more separated nucleic acid sequences from separate sources Son.Therefore, construct is that two or more nucleic acid sequences of wherein separate sources are assembled into chimeric point of single nucleic acid molecule Son and including such any construct, the construct contain (1) existing nucleic acid sequence not together in nature (that is, at least one of nucleotide sequence is heterologous relative at least one of other nucleotide sequences), including in nature It is existing not together to adjust sequence and coded sequence, or (2) encode the portion of the functional RNA molecule or protein that do not adjoin naturally The sequence divided, or the part that do not adjoin naturally of (3) promoter.Representative construct includes being derived from any source, Neng Goujin Row genome conformity or any recombinant nucleic acid molecules independently replicated, such as plasmid, clay, virus, autonomous science polynucleotides Molecule, bacteriophage or line style or cyclic single strand or double-stranded DNA or RNA nucleic acid molecules, including wherein one or more nucleic acid molecules The nucleic acid molecules effectively connected.Construct of the invention is usually by necessity member comprising instructing purpose nucleic acid sequence to express Part, the purpose nucleic acid sequence are also contained in construct, e.g., for example, target nucleic acid sequence or instrumentality nucleic acid sequence.This class component It may include control element, the promoter of (thus instructing its transcription) such as effectively connect with purpose nucleic acid sequence, and often also Including poly-adenosine sequence.Within the scope of certain embodiments of the present invention, construct be may include in carrier inside.Except structure It builds except the component of body, carrier can also be for example comprising one or more selected markers, one or more replication orgin (such as protokaryons Replication orgin and eukaryon replication orgin), at least one multiple cloning sites and/or promote construct stable integration enter host cell base Because of the element of group.Two or more constructs may include in single nucleic acid molecule (such as single carrier) inside, or can contain It is internal in two or more nucleic acid molecules (such as carriers of two or more difference) respectively." expression construct " is usually wrapped The control sequence effectively being connect containing at least one with purpose nucleotide sequence.In this way, for example, being mentioned in expression construct For the promoter effectively being connect with nucleotides sequence to be expressed, for the expression in biology or part thereof (including host cell). In order to implement the present invention, it is used to prepare and the use of the conventional composition and method of construct and host cell is those skilled in the art Known to member, see, for example, Molecular Cloning:A Laboratory Manual, the 3rd edition volume 1, the 2nd and the 3rd, J.F.Sambrook, D.W.Russell and N.Irwin, Cold Spring Harbor Laboratory Press, 2000.

" corresponding to " or " with ... it is corresponding " mean to show obvious sequence similarity or same with reference amino acid sequence The amino acid sequence of one property.In general, amino acid sequence at least part with reference amino acid sequence is shown at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even more Up to 100% sequence similarity or identity.

As used herein, term " structural domain " refers to that the part of molecule or structure, the part share common physical chemistry Feature e.g., but is not limited to hydrophobicity, polarity, spherical and helix domain or attribute, as ligand binding, film fusion, signal turn It leads, Premeabilisation of cells etc..In general, structural domain has the protein structure folded, the protein structure of the folding, which has, retains it The ability of tertiary structure, it is unrelated with protein rest part.In general, structural domain is responsible for the standalone feature attribute of protein, and In many cases, it can be added, removed or be transferred to other protein, while the residue of protein and/or structural domain Partial function is not lost.Structural domain can with multiple regions or part thereof and deposit；Structural domain can also include the difference of molecule Discontinuity zone.The example of protein domain includes but is not limited to cellular localization domain or extracellular localization domain (example Such as, signal peptide；SP), immunoglobulin (Ig) structural domain, film fusion are (for example, fusogenic peptide；FP) structural domain, extracellular domain, film Proximal end perimeter (MPER) structural domain, cross-film (TM) structural domain and cytoplasm (C) structural domain.

In excitation for the immune response or treatment or prevention disease of enveloped virus fusion protein or fusion protein complex Or in the context of symptom, " effective quantity " mean in single dose or as certain series part, to there is the individual of demand to apply certain The substance of a amount is effective to this excitation, treatment or prevention.Effective quantity will depend on it is to be treated individual health and physical condition, The taxon of individual to be treated, the preparation of composition, medical conditions are assessed and other are changed in relation to factor.It is expected that the amount will be fallen In the relatively wide in range range that can be determined by routine test.

Term " endogenous " refers to the polypeptide or part thereof for existing and/or naturally expressing in host organism or its cell interior. For example, " endogenous " extracellular domain polypeptide or part thereof refers to the extracellular of the envelope fusion protein naturally expressed in enveloped virus The part of Domain Polypeptide or the extracellular domain.

As used herein, term " area endogenous HRA " refers to such area HRA, in I class extracellular domain polypeptide, In It is deposited at the position substantially the same with the amino acid sequence area Zhong HRA of fusion protein precursor forms of naturally occurring fusion protein In.The about amino acid position in the area endogenous HRA of I class fusion protein non-limitative example is listed in table 3.

The about position in the area HRA in the selected I class fusion protein of table 3

As used herein, term " area endogenous HRB " refers to such area HRB, in I class extracellular domain polypeptide, In It is deposited at the position substantially the same with the amino acid sequence area Zhong HRB of fusion protein precursor forms of naturally occurring fusion protein In.The about amino acid position in the area endogenous HRB of I class fusion protein non-limitative example is listed in table 4.

The about position in the area HRB in the selected I class fusion protein of table 4

Term " endogenous generation " refer to the expression of biological amplifying nucleic acid and the expression product of biological amplifying nucleic acid it is related generate and/or Secretion.In a particular embodiment, biology is many cells (for example, vertebrate, preferably mammal, more preferable kobold Long class such as people) and nucleic acid expressed in the cell or tissue of multicellular organism.

As used herein, " enveloped virus merge with extracellular domain polypeptide " refers to such polypeptide, containing envelope virus at The virion surface expose portion of ripe fusion protein with and without signal peptide, but is the absence of naturally occurring enveloped virus and melts The transmembrane domain and cytoplasm tail of hop protein.

Term " epitope " and " antigenic determinant " be used to refer to interchangeably herein can in conjunction with antibody specificity (this Class epitope be often referred to as " B cell epitope ") or to T cell receptor present major histocompatibility complex (MHC) albumen (example Such as, I class or II class) (this kind of epitope be often referred to as " t cell epitope ") antigen, refer generally to protein determinant.In B cell table In the case that position is peptide or polypeptide, it generally comprises three or more amino acid, and typically at least 5 and more generally at least 8 to 10 amino acid.Amino acid can be amino acid residue (the often referred to as adjacent peptide sequence adjoined in the primary structure of polypeptide Column), or can become to be spatially near (often referred to as non-adjacent peptide sequence) in the protein of folding.T cell epitope can In conjunction with MHC I class or MHC II class molecule.Generally, in conjunction with long 8 to 11 amino acid of the t cell epitope of MHC I class.II class Molecule combination can grow 10 to 30 residues or longer peptide, and optimum length is 12 to 16 residues.It can predict simultaneously sample plot Ability (Dimitrov et al., 2010.Bioinformatics 26 of the confirmation presumption property t cell epitope in conjunction with MHC molecule (16):2066-8)。

Term " helical bundle " refers to multiple such foldings, so that spiral is substantially parallel to each other or antiparallel peptide spiral.It is double Helical bundle tool is there are two so folding, so that they are substantially parallel to each other or antiparallel spiral.Equally, Six helix bundle has Six so fold, so that they are substantially parallel to each other or antiparallel spiral." substantially parallel or antiparallel " mean as This is folded, thus the spiral that the side chain of spiral can interact with each other.For example, the hydrophobic side chains of spiral can be each other Effect is to form hydrophobic core.

Term " heterologous " as used herein refers to any protein portion, and sequence selects in such a manner, thus this The product of a sequence and extracellular domain peptide fusion has the precursor or mature form with wild type enveloped virus fusion protein Different sequences.

Term " host " refers to any biological or its cell that construct of the present invention can be introduced to it, no matter eukaryon or original Core, particularly, wherein there is the host of RNA silencing.In specific embodiments, term " host " refers to eucaryote, including Unicellular eukaryote such as yeast and fungi and multi-celled eukaryotes such as animal, non-limitative example include invertebrate (for example, insect, coelenterate, echinoderm, nematode etc.)；Eukaryon parasitic body is (for example, malaria parasitic body, such as plasmodium falciparum (Plasmodium falciparum), worm etc.)；Vertebrate is (for example, fish, amphibian animal, reptiles, bird, lactation are dynamic Object)；With mammal (for example, rodent, primate such as mankind and non-human primates).Therefore, term " host cell " is appropriate Cover this kind of Eukaryotic cell and derived from this kind of Eukaryotic cell line in ground.

It includes the Interpolymer Association for referring to any interaction, reaction or other forms that " immune interaction ", which is mentioned above, Effect and especially wherein molecule first is that or simulation Immune System Components the case where.

As used herein, term " immunogenic composition " or " immunogenic formulation " refer to when being applied to vertebrate, outstanding When its animal such as mammal, the prepared product of immune response will be induced.

" connector " mean to connect two molecules and often playing the role of two molecules are placed in required configuration molecule Or molecule group (such as monomer or polymer).

As used herein, term " meta-stable ", such as background in protein (for example, enveloped virus extracellular domain polypeptide) Under, refer to the unstable conformational state for being quickly converted to more stable conformational state when conditions are changing.For example, before merging under form Enveloped virus fusion protein is in unstable meta-stable conformation, and when for example, being blended in host cell, is transformed into more steady Conformation after fixed fusion.

As used herein, term " part " refers to that the part of molecule, the part can be intramolecule and be responsible for the molecule Characteristic chemical, a functional group of biology and/or medicine attribute, one group of functional group and/or one group of specific atom.

Term " neutrality antigen binding molecules " refers to such antigen binding molecules, with target molecule or ligand binding or with Interaction and prevent target antigen and associativity gametophyte such as receptor or Binding Capacity or association, thus suspend otherwise this by because The interaction of molecule and the biologically generated.In the present case, neutrality antigen binding molecules suitably with Meta-stable or the enveloped virus fusion protein for merging preceding form associate and preferably interfere or reduce fusion protein and cell membrane Combination and/or fusion.

With at least in principle by the polymer of the unrestricted monomer composition of number on the contrary, term " oligomer " refers to by being more than One but a limited number of monomeric unit composition molecule.Oligomer includes but is not limited to dimer, tripolymer, the tetramer, five Aggressiveness, six aggressiveness, heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness etc..Oligomer can be non-total by macromolecular (such as protein) The macromolecule complex that valence bonds together to form.Under this meaning, homotype oligomer will be formed by identical molecule, and on the contrary, different Source oligomer will be by least two different molecular compositions.In a particular embodiment, oligomer of the invention is by more than three The trimeric polypeptide complex of peptide subunit composition.In these embodiments, trimeric polypeptide can be by three phase homopolypeptides " the homotrimer complex of polypeptides " of subunit composition or by different three polypeptide moieties of wherein at least one subunit polypeptide " the heterotrimer complex of polypeptides " of composition." polypeptide moiety " is to combine that form trimeric polypeptide multiple with two other polypeptide moieties Fit single amino acid chain or monomer.

As used herein, term " effectively connection " or " effectively connecting " refer to juxtaposition, wherein at the component so described In the relationship for allowing them to function in a manner of its expection.For example, with purpose nucleotide sequence (for example, coded sequence and/ Or non-coding sequence) " effectively connection " adjusting sequence (for example, promoter) control sequence relative to purpose nucleotide sequence Positioning and/or orientation are to allow aim sequence to express under conditions of being compatible to control sequence.Control sequence need not be with purpose core Nucleotide sequence is adjacent, as long as they play the effect for instructing it to express.Thus, for example, interleave non-coding sequence (for example, The sequence do not translated but transcribed) it can reside between promoter and coded sequence, and the promoter sequence can still be considered as " effectively being connect with coded sequence ".Similarly, enveloped virus fusion extracellular domain polypeptide and heterologous structural stabilize part " effectively connection " covers the positioning and/or orientation of structure stabilization part, so that the complementary area HR1 and the area HR2 be allowed to be suitable for They associate each other under conditions of (for example, in aqueous solution) associating, to form antiparallel double helix beam.

The term " patient ", " subject ", " host " or " individual " being used interchangeably herein refers to that it needs to treat or prevent Any subject, in particular to vertebrate subject, and even more in particular to mammalian subject.Fall within this hair Suitable vertebrate in bright range includes but is not limited to any member of subphylum chordata, including primate (for example, people, Monkey and ape, and including monkey species, such as from Macaca (Macaca) (for example, machin class, such as machin (Macaca Fascicularis) and/or Henghe monkey class (macaque (Macaca mulatta))) and baboon (globefish tail baboon (Papio Ursinus)) and marmoset class (species for coming from marmoset category (Callithrix)), Squirrel monkey (come from Saimiri (Saimiri) Species) and the willow monkey class species of (come from Chinese tamarisk Lagothrix (Saguinus)) and ape species, such as chimpanzee class (chimpanzee (Pan troglodytes))), rodent (for example, mouse rat, cavy), Lagomorpha (for example, rabbit, hare), ox (for example, Ox), sheep (for example, sheep), goat (for example, goat), pig (for example, pig), horse (for example, horse), dog (for example, dog), cat (for example, Cat), birds (for example, chicken, turkey, duck, goose, pet bird such as canary, parrot etc.), marine mammal (for example, dolphin, Whale), reptiles (snake, frog, lizard etc.) and fish.Preferred subject be need to excite for enveloped virus fusion protein or The people of the immune response of fusion protein complex.It is to be understood, however, that term above-mentioned does not show that there are symptoms.

" pharmaceutical acceptable carrier " mean can to animal, preferably mammal (including people) is topically or systemically applied when pacify Solid-state or liquid fillers, diluent or the encapsulating substance entirely used.Representative pharmaceutical acceptable carrier include any and whole solvents, Decentralized medium, coating, surfactant, antioxidant, preservative (for example, antibacterial agent, antifungal agent), isotonic agent, absorption Delayed-action activator, salt, preservative, drug, drug stabilizing agent, gel, adhesive, excipient, disintegrating agent, lubricant, sweetener, flavoring Agent (flavoring agents), dyestuff, this kind of analog material and combinations thereof, (example as known to those of ordinary skill in the art Such as, referring to Remington ' s Pharmaceutical Sciences, the 18th edition, Mack Printing Company, 1990, The 1289-1329 pages, the document is incorporated herein by reference).In addition to any conventional carrier is incompatible with effective component In the case where except, contemplate purposes of the carrier in pharmaceutical composition.

Term " polynucleotides " or " nucleic acid " as used herein refer to mRNA, RNA, cRNA, cDNA or DNA.The term one As refer to length at least ten base polymer form nucleotide (the two of ribonucleotide or deoxynucleotide or modified forms Seed type nucleotide it is any).The term includes single-stranded and double-stranded form DNA.

" polypeptide ", " peptide ", " protein " and " protein molecule " is used to refer to residual comprising amino acid interchangeably herein The polymer of base and its variant and synthetic analog or the molecule being made from it.Therefore, these terms are suitable for one of them Or more amino acid is that (chemistry such as corresponding naturally occurring amino acid is similar for the non-naturally occurring amino acid of synthesis Object) amino acid polymer and naturally occurring amino acid polymer.

As used herein, " conformation after fusion " of term enveloped virus fusion protein refers to the knot of enveloped virus fusion protein Structure is in terminal conformation (that is, being formed at the end of fusion process) and is the state of energy favorable.The conformation after fusion In, make fusion protein fusogenic peptide or ring and fusion protein transmembrane domain close to.Promotion forms the specific structure of hairpin structure Property element according to the classification of envelope fusion protein change.For example, conformation is after the fusion of I class fusion protein with each I class fusion protein The area endogenous HRA and the area endogenous HRB between interaction characterized by forming hairpin structure, the hairpin structure is to wrap Six helix bundle containing three areas endogenous HRB and three areas endogenous HRA is characterized.Alternatively, the fusion of Group III fusion protein Conformation between internal fusion ring and the C-terminal transmembrane region for promoting hairpin structure to be formed characterized by interacting afterwards.Pass through Electron microscopy and/or X-ray crystallography measure conformation after the fusion of each virus amalgamation protein, in negative staining electron micrograph In when checking and/or according to epitope before fusion is lacked, can recognize this kind of structure easily.

As used herein, " conformation before merging " of term enveloped virus fusion protein refers to the knot of enveloped virus fusion protein Structure, the structure is in meta-stable conformation, and (that is, in semi-stability conformation, the semi-stability conformation is not energy favorable Terminal conformation) and when being suitable for triggering can occurred conformation reset become terminal fusion after conformation.Generally, virus amalgamation protein Fusion before conformation contain be located at merge before inside conformation and cannot be hydrophobic with viromembrane or host cell membrane interaction Property sequence, referred to as fusogenic peptide or fusion ring.Once triggering, in this hydrophobic signal sequence Insertion Into Host Cell film and fusion protein Collapse to hair clip sample conformation after merging.Conformation is changed according to the classification of envelope fusion protein before the fusion of virus amalgamation protein.Often A classification is characterized by noninteracting structural member, and the structural member is then with structure after the advantageous fusion of energy As association.For example, before the fusion of I class fusion protein the respective fusion protein of conformation dependence Yu Buyu tripolymer endogenous HRB The area endogenous HRA of area's interaction, because without allowing the hairpin structure characterized by Six helix bundle to be formed.Alternatively, Group III Fusion ring interaction before the fusion of fusion protein at the C-terminal region of the respective fusion protein of conformation dependence Yu Buyu tripolymer Central alpha-helix coiled coil, because without allow hairpin structure formed.Electron microscopy and/or X-ray knot are passed through Crystalline substance learns conformation before the fusion for measuring each virus amalgamation protein, when being checked in negative staining electron micrograph and/or according to fusion after Epitope before the fusion being not present in conformation can recognize this kind of structure easily.

" regulating element ", " adjusting sequence ", control element ", " control sequence " etc. are used to refer to this interchangeably herein The nucleotide sequence of sample, be located at upstream of coding sequence (5' non-coding sequence), internal or downstream (3' non-coding sequence) and Directly or indirectly influence transcription, RNA processing or the stability or translation of engaged coded sequence.Regulating element includes enhancing Son, promoter, translation leader sequence sequence, introne, Rep recognition component, intergenic region and polyadenylation signal sequence.It Include native sequences and composition sequence and the sequence that can be composition sequence and native sequences combination.

Term " replicon " refers to the independent unit performance that portion in the cell is replicated as polynucleotides, that is, can its from The lower any genetic elements replicated of body control, for example, plasmid, chromosome, virus, clay etc..

" self-assembly " refers to the process of the spontaneous assembly of higher structure, dependent on higher structure component (for example, molecule) that This naturally attracts.It is generally occurred by the random motion of molecule and is formed based on size, shape, composition or chemical attribute Key.

Term " sequence identity " as used herein refers to that sequence is based on nucleotide vs nucleotide or based on amino acid to ammonia Base acid identical degree within the scope of comparison window.Therefore, " percent sequence identities " are calculated in the following manner: compared Compare the sequence of two best alignments in window ranges；Determine wherein identical nucleic acid base (for example, A, T, C, G, I) or identical ammonia Base acid residue is (for example, Ala, Pro, Ser, Thr；Gly,Val,Leu,Ile,Phe,Tyr,Trp,Lys Arg,His,Asp, Glu,Asn；Gln, Cys and Met) number of position that occurs in the two sequences to generate the number of matching position, will match It is same that the number of position divided by the total number of positions in comparison window (that is, window size) and by result generates sequence multiplied by 100 One property percentage.This invention contemplates overall length IL-22 polypeptides and its bioactive fragment in the method for the present invention and system Purposes.Generally, the bioactive fragment of overall length IL-22 polypeptide can participate in interacting, for example, intramolecular or intermolecular mutual Effect.

" similitude " refers to the percentage of amino acid that is identical or constituting preservative replacement defined in Tables 1 and 2 as above. Sequence comparison program such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12:387-395) can be used Measure similitude.In this manner, it may be possible to by being inserted into vacancy to comparison result, comparison length and sequence those of is quoted herein Sequence that is similar or being different in essence is arranged, such as this kind of vacancy is determined by the comparison algorithm that GAP is used.

It include: " reference sequences ", " ratio for describing the term of sequence relation between two or more polynucleotides or polypeptide Compared with window ", " sequence identity ", " percent sequence identities " and " Substantial identity "." reference sequences " it is long at least 12 but Often 15 to 18 and frequent at least 25 monomer units, including nucleotide and amino acid residue.Because two polynucleotides can To respectively contain (1) similar sequence (that is, only a part of complete polynucleotide sequence) and (2) between two polynucleotides The divergent sequence between two polynucleotides, so general by comparing two polynucleotides in " comparison window " range Sequence, the sequence carried out between two or more polynucleotides compare, to identify and compare the part with sequence similarity Region." comparison window " refer to at least six, normally about 50 to about 100, more typically from about 100 to about 150 close positions Conceptual section, wherein by a sequence with the reference sequences with identical close position number in the two sequences of best alignment After compare.Such as compared with reference sequences (not including addition or missing), comparison window may include about 20% or less addition Or missing (that is, vacancy) is with two sequences of best alignment.Algorithm (Wisconsin Genetics Software can be passed through In Package Release 7.0, Genetics Computer Group, 575Science Drive Madison, WI, USA GAP, BESTFIT, FASTA and TFASTA) computerization execute or by inspection and with any one in selected various methods The optimal comparison result (that is, generating highest homology percentage within the scope of comparison window) of generation, implements for being aligned comparison window The optimal comparison of the sequence of mouth.It can also be with reference to such as such as Altschul et al., 1997, Nucl.Acids Res.25:3389 public affairs The blast program family opened.Can be in Ausubel et al., " Current Protocols in Molecular Biology ", John Wiley&Sons Inc, 1994-1998 find being discussed in detail for sequence analysis in the 15th chapter Unit the 19.3rd.

As used herein, term " single-stranded " refers to the molecule comprising the amino acid monomer by peptide bond linearly connected.

As used herein, " small molecule " refer to have less than 3 kilodaltons (kDa) and generally less than 1.5 kilodaltons and The more preferably less than about composition of the molecular weight of 1 kilodalton.Small molecule can be nucleic acid, peptide, polypeptide, peptide mimics, sugar Class, lipid or other organic (carbon containing) or inorganic molecules.Such as skilled artisans will appreciate that, it is based on this specification, Ke Yiyong Identification adjusts any one extensive chemical mixture of screening and/or biology of the measuring method of the present invention of the compound of bioactivity Mixture (often fungal extract, bacterial extract or algae extract) library." small organic molecule " is that have less than 3,000 Er Dun, less than 1.5 kilodaltons or be even less than about 1kDa molecular weight organic compound (or with inorganic compound (for example, Metal) compound organic compound).

As used herein, pharmacodynamics effect and/or physiological effect needed for the fingers such as term " treatment ", " processing " obtain.The effect It should can be preventative for preventing disease or its symptom completely or partially and/or just can partially or completely cure disease And/or it is attributed to for the side effect of the disease be therapeutic.As used herein, " treatment " is covered in mammal, especially Any treatment of disease in its mankind, and include: that (a) prevents disease from may tend to that the disease but not yet diagnosis trouble occurs Occur in the subject for having this disease；(b) inhibit this disease, that is, prevent its progress；(c) mitigate the disease, that is, draw Play the disease regression.

Term " wild type ", " natural " and " naturally occurring " is used to refer to gene interchangeably herein or gene produces Object, the feature with the gene or gene product when being separated from naturally occurring source.Wild type, natural or naturally occurring base Cause or gene product (for example, polypeptide) are that the gene or gene production are most-frequently observed and be therefore arbitrarily designated as in group That of " normal " or " wild type " form of object.

Unless expressly stated otherwise, in the case of otherwise having made necessary amendment, each embodiment as described herein should be applicable in In every kind and each embodiment.

2. chimeric polyeptides

The enveloped virus fusion protein that the present invention is based in part under servostabilization or the preceding conformation of ' clamp ' fusion is extracellular The new strategy of Domain Polypeptide.This ' molecule clamp ' strategy merging using structure stabilization part and extracellular domain polypeptide Or bonding is to form chimeric polyeptides.Structure stabilization part is usually the single chain polypeptide for including complementary heptapeptide repetitive sequence, institute State heptapeptide repetitive sequence lack with the complementarity of extracellular domain polypeptide and therefore associate each other to preference rather than with extracellular knot The structural member of structure domain polypeptide is associated.Complementary heptapeptide repetitive sequence is under conditions of being suitable for them and associating (for example, water-soluble In liquid) each other association result in antiparallel double helix beam and formed, the antiparallel double helix beam inhibits extracellular domain polypeptide It is rearranged into conformation after merging.The double helix beam of structure stabilization part can with trimerizing to form highly stable Six helix bundle, Therefore allow chimeric polyeptides self-assembly to form artificial enveloped virus fusion protein complex.The complex so assembled can be with mould Conformation and include three kinds chimeric more characterized by Six helix bundle before the fusion of quasi- enveloped virus native fusion proteins complex Peptide, the Six helix bundle are formed by the coiled-coiled structure of the respective structure stabilization part of chimeric polyeptides.

2.1 structure stabilization parts

The present inventor constructs the single chain polypeptide part comprising complementary heptapeptide repetitive sequence, the heptapeptide repetitive sequence folding Antiparallel conformation is built up, the anti-of the enveloped virus extracellular domain stabilizing polypeptides in the preceding conformation of fusion for making effectively to connect is formed Parallel double helix beam.Double helix beam is properly formed coiled-coiled structure.Coiled-coil folds appear in a plurality of types of albumen In matter, including motor protein, DNA binding protein, exoprotein and virus amalgamation protein (see, for example, Burkhard et al., 2001.Trends Cell Biol 11:82-88).Coiled coil functionally (assemble, oligomerization) as folding by characterization Motif, that is, form the coiled-coiled structure for driving different proteins chain noncovalent associations in many cases.Coiled coil is The alpha-helix assembly for 2-, 3-, 4- or 5- chain arranged by parallel, antiparallel or mixed topology structure is characterized as in structure (for example, with reference to Lupas, 1996.Trends Biochem Sci 21:375-382).In general, spiral presses left or right hand mode Slightly about package each other (winding, winding), referred to as supercoil.It should be appreciated that double helix beam of the invention, which is usually formed, to be had Trimerizing is to form the coiled-coiled structure of the strong tendency of six highly stable helical coil-coil beams.

2.1.1 heptapeptide repetitive sequence

Alpha-helix coiled coil is characterized in its amino acid sequence level, is characterized in that, each spiral is by a series of Heptapeptide repetitive sequence is constituted.One heptapeptide repetitive sequence (septuple body unit, septuple body) be can be encoded to hpphppp and its In each ' h ' represent hydrophobic residue and each ' p ' is 7 residue sequence motifs of polar residues.Once in a while, p is observed in the position h Residue, and vice versa.Heptapeptide repetitive sequence is also often according to pattern a-b-c-d-e-f-g (abcdefg) or d-e-f-g- A-b-c (defgabc) coding, in this case, mark ' a ' to ' g ' refer to conventional the seven of the common amino acid type observed Weight position.According to convention, mark ' a ' and ' d ' refers to the position of core residue in coiled coil (center, embedding residue).In core The common amino acid type that a- and d- is observed position is hydrophobic amino acid residues type；It is (non-core in whole other positions Position), advantageously observe polarity (hydrophily) residue type.Therefore, conventional septuple body pattern ' hpphppp ' and pattern are remembered (' hppphpp ' pattern is matched with pattern mark ' defgabc ', and this mark is used to start from the position d- for number ' abcdefg ' matching The coiled coil of hydrophobic residue).Heptapeptide repetitive sequence area of the invention include in each alpha-helix coiled-coiled structure extremely Few 2 and suitably 3 or more continuous (uninterrupted) heptapeptide repetitive sequences.The continuous heptapeptide of each series in one spiral Repetitive sequence is referred to as ' heptapeptide repetitive sequence ' (HRS).If can get, it is based preferably on three-dimensional (3-D) structure of measuring Determine the beginning and end of heptapeptide repetitive sequence.If 3-D structure is unavailable, it is based on real amino acid sequence pair (hpphppp)_nOr (hppphpp)_nThe Optimal coverage of pattern determines beginning and end, wherein ' h ' and ' p ' of heptapeptide repetitive sequence Hydrophobic residue and polar residues are respectively referred to, and wherein ' n ' is equal to or greater than 2 number.Each heptapeptide repetitive sequence Beginning and end is taken as first hydrophobic residue and most end hydrophobic residue at the position Chu Huod- of the position a respectively.Conventional H is residual Base is preferably chosen from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, paddy Glutamine, threonine, serine and alanine are more preferably selected from valine, isoleucine, leucine and methionine, and And most preferably isoleucine.Conventional p- residue is preferably chosen from glycine, alanine, cysteine, serine, Soviet Union's ammonia Acid, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine.If this method does not allow Unambiguously ownership amino acid residue can apply more dedicated analysis method, such as Lupas et al. to heptapeptide repetitive sequence COILS method (1991.Science 252:1162-1164；

http://www.russell.embl-heidelberg.de/cgi-bin/coils-svr.pl)。

In specific embodiments, each heptapeptide repetitive sequence area (HR1, HR2) is independently with the n times of amino acid classes It repeats 7 residue patterns to be characterized, the pattern is expressed as (a-b-c-d-e-f-g-)_nOr (d-e-f-g-a-b-c-)_n, such as in WO 2010/066740 for example described in like that, the content of the document is completely incorporated herein by reference, and wherein pattern is wanted Plain ' a ' to ' g ' refer to its be in the conventional septuple position of amino acid classes and n be equal to or greater than 2 number, and conventional seven At least 50% (or at least 51% whole whole number percentages at least 99% and between them) of weight position ' a ' and ' d ' are by dredging Aqueous amino acid classes occupy and conventional septuple position ' b ', ' c ', ' e ', ' f ' and ' g ' at least 50% (or at least 51% arrive At least 99% and whole whole number percentages between them) occupied by hydrophilic amino acid type, resulting hydrophobic amino acid Distribution between type and hydrophilic amino acid type makes it possible to identify heptapeptide repetitive sequence area.In a particular embodiment, The conventional septuple position ' a ' of at least 50%, 70%, 90% or 100% and ' d ' are selected from valine, isoleucine, bright by amino acid Propylhomoserin, methionine or its non-natural derivative occupy.Due to the latter, amino acid corresponds to more standard (more frequently observing) Coiled coil core residue.In other embodiments, the conventional septuple position ' a ' of at least 50%, 70%, 90% or 100% ' d ' is occupied by isoleucine.In some embodiments, the conventional septuple position of at least 50%, 70%, 90% or 100% ' b ', ' c ', ' e ', ' f ' and ' g ' is by being selected from glycine, alanine, cysteine, serine, threonine, histidine, asparagus fern acyl Amine, aspartic acid, glutamine, glutamic acid, lysine, arginine or its non-natural derivative amino acid occupy.This In the illustrative example of type, the area HR1 and the area HR2 include following sequence, is made from it or consisting essentially of: IEEIQKQIAAIQKQIAAIQKQIYRM[SEQ ID NO:1]

In specific embodiments, the area HR1 and the area HR2 of structure stabilization part (also referred to herein as " SSM ") include I At least one endogenous heptapeptide repetitive sequence of class enveloped virus fusion protein.Suitably, the area HR1 and the area HR2 respectively mainly by The complementary area HRA and the area HRB of one or more I class enveloped virus fusion proteins is formed.HRA region amino acid sequence and the area HRB Amino acid sequence can be derived from identical I class enveloped virus fusion protein.Alternatively, they can be derived from different I classes Enveloped virus fusion protein.In representative example, the area HR1 and the area the HR2 area HRA and the area HRB independently selected from following virus: Orthomyxovirus (for example, Flu-A (Inf A), influenza B (Inf B), influenza virus C (Inf C)), paramyxovirus (for example, morbilli (MeV), rinderpest virus (RPV), canine distemper virus (CDV), RSV, human metapneumovirus (HMPV), parainfluenza virus (PIV), mumps virus (MuV), Hendra virus (HeV), Nipah virus (NiV), newcastle disease virus (NDV)), reverse transcription disease Malicious (for example, 1 type of human T cell leukemia virus (HTLV-1), HTLV-2, HTLV-3, HIV-1, HIV-2), filamentous form virus (example Such as, Ebola virus (EBOV), including Zaire (ZEBOV) strain, Reston (REBOV) strain and the Sudan (SEBOV) strain, Marburg Viral (MARV)), Arenavirus is (for example, Lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), Jun í n is viral (JUNV)) and coronavirus (for example, human corona virus (HCoV) including HCoV 229E, HCoV OC43, HCoV HKU1, HCoV EMC, people convex grand viral (HToV), Middle East breath syndrome virus (MERS-CoV), serious acute respiratory are comprehensive Levy viral (SARS-CoV)).

Exemplary HRA region amino acid sequence includes but is not limited to those of in table 5:

The HRA region sequence of the selected I class fusion protein of table 5

Exemplary HRB region amino acid sequence includes but is not limited to those of in table 6:

The HRB region sequence of the selected I class fusion protein of table 6

The area HR1 and the area HR2 can gather together to be formed thermodynamically stable and embody I viroid fusion protein and melt The oligomer of conformation after conjunction, usually by three areas HR1 and three HR2 district's groups at six aggressiveness.There is strong oligomerization herein Change the tendentious area HR1 and the area HR2 and is referred to as " complementary " heptapeptide repetitive sequence area.This kind of heptapeptide repetitive sequence area it is non-limiting Example is those of to list in table 7.

In specific embodiments, the structure stabilization part comprising one or both in heptapeptide repetitive sequence area includes Immune silencing suppresses part, and the part inhibits the excitation or generation of the immune response for structure stabilization part, special It is not such when being folded into antiparallel double helix beam.These embodiments are advantageous, because they can permit generation more Strong or enhancing the immune response for extracellular domain polypeptide or its complex.Immune silencing moiety can be by glycosylating Enzyme, especially glycosyl transferase specific recognition and glycosylated glycosylation site.Glycosylation can be N connection or O connection.N- connection refers to The engagement of saccharide part and asparagine residue side chain.Tripeptide sequence N-X-S and N-X-T, wherein X is any amino in addition to p Acid is the identification sequence that saccharide part is engaged with asparagine side chain enzymatic, and these sequences are commonly referred to as ' glycosylation site '.O- Linked glycosylation refers to following sugar: one of N- acetylgalactosamine, galactolipin or xylose and hydroxyamino acid, most common and serine or Soviet Union The engagement of propylhomoserin, but 5-OxoPro or 5- hydroxylysine also can be used.Immune silencing moiety can be inserted to comprising seven In peptide repetitive sequence area in the structure stabilization part of one or both.

In other embodiments, using widened genetic code, non-natural or unnatural amino acid can be incorporated to seven In one of peptide repetitive sequence area or both.It is orthogonal right using tyrosyl-tRNA/ aminoacyl-tRNA synthetase in purpose site And nonsense codon, unnatural amino acid is incorporated in destination locations in a manner of biosynthesis.By non-natural or non-natural amino Acid is supplied to the cell for expressing the construct that can therefrom express chimeric polyeptides from external source, and this strategy can be incorporated to tool Have a side chain of wide range of types physical attribute, including but not limited to chemical crosslinking group (for example, azide or halogenated alkane), can Marker (for example, fluorescence or radioactivity) and photosensitive group are tracked, so that time control modification is possibly realized.For these non-naturals Amino acid, each section can be connect to provide advantageous attribute by chemically adding with structure stabilization some covalent.

The complementary heptapeptide repetitive sequence region sequence of table 7

Other embodiments may include combining with any possibility of the above-mentioned example of structure stabilization some covalent connection Or non-natural additional chemical addition.

It is optionally possible to one or more additional cysteine residues are inserted into the area HR1 and/or HR2, to form two sulphur Key and further rock-steady structure stabilize antiparallel, the alpha-helix coiled coil of part.

2.1.2 connector

Structure stabilization part of the invention suitably include interval heptapeptide repetitive sequence area (also referred to herein as HR1 and HR2 connector).Connector generally comprises any amino acid residue that cannot unambiguously belong to heptapeptide repetitive sequence.Connector It is frequently used for protein engineering field, to interconnect different functional units, such as generating derived from antibody variable light chain (VL) and single chain variable fragment (scFv) construct of variable heavy chain (VH).In the solution, they are usually in Conformational flexibility, and It suitably and advantageously include polar amino acid residues type.The amino acid of (frequently using) common in flexible joint is serine And glycine.Less preferably, flexible joint can also include alanine, threonine and proline.Therefore, structure stabilization portion The interleaving property joint divided preferably is flexibility in conformation, and (without hindrance) association of the relaxation to ensure HR1 and HR2 is suitably to take The double helix beam of alpha-helix coiled-coiled structure.It will be that technical staff is aobvious and easy for the suitable linkers in polypeptide contemplated herein See, and usually can be any connector that this field is used to connect amino acid sequence, as long as connector is flexible in structure , that is, mean their permissions and the suitably not assembly of the characteristic double helix binding structure of structural damage stabilisation part.

Those of ordinary skill in the art will determine best connector, optionally after carrying out a limited number of routine experiment It determines.Interleaving property joint is suitably usually made of at least one amino acid residue and usually by least two amino acid residue group At amino acid sequence, be about 100 amino acid residues for the non-key upper limit selected by convenient reason.Specifically implementing In scheme, connector is by about 1 to about 50 amino acid residue or about 50 to about 100 amino acid residues, normally about 1 to about 40 Amino acid residue, general about 1 to about 30 amino acid residue composition.In non-limitative example, connector has and connect I class packet The amino acid of the about the same number of number of the amino acid in the area the HRA and area HRB of film virus amalgamation protein complementation.Specific non- In restricted embodiment, at least 50% amino acid residue of joint sequence is selected from proline, glycine and serine.At other In non-limiting embodiments, at least 60%, such as at least 70%, such as such as 80% and more specifically 90% ammonia of joint sequence Base acid residue is selected from proline, glycine and serine.In other specific embodiments, joint sequence is substantially by polarity Amino acid residue composition；In this kind of specific embodiment, at least 50%, such as at least 60%, such as such as the 70% of joint sequence Or 80% and more specifically 90% or until 100% amino acid residue be selected from glycine, serine, threonine, alanine, dried meat Propylhomoserin, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine.In specific embodiment In, joint sequence may include [GGSG]_nGG、[GGGGS]_n、[GGGGG]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、 [GGGCGGGG]_n, wherein n be from 1 to 10, suitably from 1 to 5, more suitably from integer of 1 to 3.

Heptapeptide repetitive sequence area separately include I class enveloped virus fusion protein complementation the area HRA and the area HRB, by its group At or consisting essentially of specific embodiment in, connector include connection the area HRA and the area HRB the naturally occurring ammonia of interleaving property Base acid sequence is made from it or consisting essentially of.The interleaving property sequence can be overall length or substantially overall length or can be with One or more parts comprising the naturally occurring amino acid sequence of interleaving property of overall length are made from it or substantially by forming.At it In his embodiment, connector lack interleave it is natural between the area HRA and the area HRB of wild type I class enveloped virus fusion protein Existing amino acid sequence.In any embodiments above, connector may include one or more non-naturally occurring amino Acid sequence.

In addition to spacer structure stabilizes the heptapeptide repetitive sequence area of part and is preferably introduced configuration flexibility to promote this Except a little antiparallel associations in region, connector can also include one or more auxiliary functional groups.For example, connector may include promotion Immunological regulation part of the purification part and/or at least one metering needle of chimeric polyeptides purifying to the immune response of chimeric polyeptides.

Purification part, which generally comprises one section, can be realized the amino acid that chimeric polyeptides are recycled by affine combination.Numerous purifying Part or ' label ' be it is known in the art, illustrative example include biotin carboxyl carrier protein label (BCCP label), Myc label (c-myc label), calmodulin label, FLAG- label, HA label, His label (hexahistidine tag, His6, 6H), maltose binding protein tag (MBP label), Nus label, chitin binding protein label (CBP label), glutathione- S- transferase label (GST label), green fluorescent protein tag (GFP label), polyglutamic acid label, amyloid beta label, Thioredoxin label, S label, Softag1, Softag3, Strep label, Streptavidin combination peptide tag (SBP label), Biotin label, Streptavidin label and V5 label.

Immunological regulation part can be introduced to connector to adjust the immune response of chimeric polyeptides or the excitation of its complex.It is this kind of Partial non-limitative example includes that silencing is immunized as described above or suppresses part, antigenic portions, including is derived from and causes a disease The relevant antigenic portions of antigenic portions or other diseases of biology, such as cancer or tumor associated antigen.Exemplary pathogenic organisms Including but not limited to virus, bacterium, fungi, parasitic body, algae and protozoon and amoeba.In a particular embodiment, antigenic Part is derived from the antigen of pathogenic virus.The schematic virus for causing disease includes but is not limited to measles virus, the popular cheek Adenositis virus, rubella virus, poliovirus, hepatitis A virus, hepatitis type B virus are (for example, GenBank accession number ) and Hepatitis C Virus (for example, GenBank accession number E06890) and other hepatitis virus, influenza virus, adenopathy E02707 Viral disease poison (for example, 4 types and 7 types), hydrophobin (for example, GenBank accession number M34678), yellow fever virus, Epstein- Barr virus and other herpesvirals for example papillomavirus, Ebola virus, influenza virus, japanese encephalitis virus (for example, GenBank accession number E07883), dengue fever virus (for example, GenBank accession number M24444), Hantaan virus, sendai virus, Respiratory syncytial virus (RSV), orthomyxovirus, vesicular stomatitis virus, Wei Sina virus, cytomegalovirus and human immunodeficiency Malicious (HIV) (for example, GenBank accession number U18552).Any suitable antigen derived from this viroid can be used for implementing this hair It is bright.For example, the schematic O retrovirus antigens derived from HIV include but is not limited to that the gene of gag, pol and env gene such as produces Object, Nef albumen, reverse transcriptase and other HIV components antigen.The illustrative example of hepatitis antigen includes but is not limited to If hepatitis type B virus S, M and L albumen, hepatitis type B virus and other hepatitis virus are (for example, A type, B-mode and hepatitis C Virus) pro-S antigen antigen.The illustrative example of influenza virus property antigen includes but is not limited to such as hemagglutinin and neuraminidase With the antigen of other influenza virus components.The illustrative example of measles toxicity antigen includes but is not limited to such as measles virus fusion The antigen of albumen and other measles virus components.The illustrative example of rubella toxicity antigen includes but is not limited to such as E1 and E2 egg White and other rubella virus components antigens；Wheel virus antigen such as VP7sc and other rotavirus components.Cytomegalovirus is anti- Former illustrative example includes but is not limited to the antigen such as envelope glycoprotein B and other cytomegalovirus antigen components.Respiratory tract The non-limitative example of syncytial virus antigen includes as RSV fusion protein, M2 albumen and other respiratory syncytial virus (RSV)s are anti- The antigen of stock blend.The illustrative example of herpes simplex keratitis antigen includes but is not limited to such as early protein, glycoprotein D immediately With the antigen of other herpes simplex keratitis antigen components.The non-limitative example of varicella virus antigen includes Such as the antigen of 9PI, gpII and other varicella virus antigen components.Japanese encephalitis virus property antigen it is unrestricted Property example include such as protein E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E and other japanese encephalitis virus property antigens Fraction antigen.The representative example of hydrophobin property antigen includes but is not limited to such as rabies glycoproteins, rabies nucleoprotein With the antigen of other hydrophobin property antigen components.The illustrative example of papilloma virus antigens includes but is not limited to L1 and L2 Capsid protein and E6/E7 antigen relevant to cervical carcinoma.Additional examples about viral antigen are referring to Fundamental Virology, second edition Fields, B.N. and Knipe, D.M., 1991, Raven Press, New York.Specifically implementing In scheme, viral antigen is the antigen corresponding with extracellular domain polypeptide of enveloped virus.In other embodiments, viral Property antigen is the antigen corresponding with extracellular domain polypeptide of different enveloped virus.

It in some embodiments, will be in one or more cancers correlations or tumor associated antigen insertion connector.This kind of antigen Including but not limited to MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weight melanoma associated antigen MAA, GD2, cancer Embryonal antigen (CEA), TAG-72, ovarian associated antigens OV-TL3 and MOV18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, (chromoma correlation is anti-by CA-50, CA-19-9, kidney neoplasms related antigen G250, EGP-40 (also referred to as EpCAM), S100 It is former), p53, tumor of prostate-related antigen (for example, PSA and PSMA), p21ras, Her2/neu, EGFR, EpCAM, VEGFR, FGFR, MUC-I, CA 125, CEA, MAGE, CD20, CD19, CD40, CD33, A3, the antigen special to A33 antibody, BrE3 are anti- Original, CD1, CD1a, CD5, CD8, CD14, CD15, CD16, CD21, CD22, CD23, CD30, CD33, CD37, CD38, CD40, CD45, CD46, CD52, CD54, CD74, CD79a, CD126, CD138, CD154, B7, Ia, Ii, HMl.24, HLA-DR (for example, HLA-DR10), NCA95, NCA90, HCG and subunit, CEA (CEACAM5), CEACAM-6, CSAp, EGP-I, EGP-2, Ba 733, KC4 antigen, KS-I antigen, KS1-4, Le-Y, MUC2, MUC3, MUC4, PlGF, ED-B fibronectin, NCA 66a-d, PAM-4 antigen, PSA, PSMA, RS5, SlOO, TAG-72, TlOl, TAG TRAIL-Rl, TRAIL-R2, p53, tenascin, pancreas Island element growth factor-1 (IGF-I), Tn antigen etc..

Comprising within a fitting antigenic portions or all parts can correspond to overall length antigen or incomplete antigen.When the portion of using When dividing antigen, the incomplete antigen may include one or more epitopes of purpose antigen, including B cell epitope and/or T cell Epitope (for example, Cytotoxic T lymphocytes (CTL) epitope and/or T helper cell (Th) epitope).The epitope of numerous antigens is text It is known or routine technology well known by persons skilled in the art can be used determine in offering.In other embodiments, connector can To include another cell-targeting part, the cell-targeting part, which can provide, arrives at the internal certain detail of immunity inoculation individual The delivering of born of the same parents' type.Aim cell group includes but is not limited to B cell, microfold cell and antigen presenting cell (APC).Subsequent In example, the targeting moiety promotes the knowledge of chimeric polyeptides or its complex to the enhancing of APC such as dendritic cell or macrophage Not.The epitope that APC presents the extracellular domain polypeptide that associated can be enhanced in this kind of targeting sequence, this can transfer produced by enhancement Immune response, including strengthening or widening for any in the antibody mediated immunity response of extracellular domain polypeptide and cellullar immunologic response The specificity of person or both.The non-limitative example of APC targeting moiety includes the ligand in conjunction with APC surface receptor, such as but not Be limited to mannose specific agglutination plain (mannose receptor), IgG Fc receptor, DC-SIGN, BDCA3 (CD141), 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptor and scavenger receptor.In specific embodiments, APC targeting moiety It is comprising sequence FYPSYHSTPQRP (Uriel et al., J.Immunol.2004 172:7425-7431) or NWYLPWLGTNDW It (Sioud et al., FASEB J 2,013 27 (8): 3272-83), is made from it or the targeting of consisting essentially of dendritic cell Part.

2.2 enveloped virus fusion proteins and extracellular domain polypeptide

Molecule clamp strategy of the invention can be used for being assembled into its a series of wild type counterparts merging preceding shape in it The extracellular domain polypeptide (including I class and Group III fusion protein) of the tripolymer of formula is stablized.Unrestricted I class fusion protein packet Include fusion protein (for example, HA albumen of influenza A virus, influenza B virus and influenza virus C), the pair of orthomyxovirus The fusion protein (for example, F protein and MeV, RPV, CDV, RSV, HMPV, PIV, MuV, HeV, NiV and NDV albumen) of myxovirus, The fusion protein (for example, envelope glycoprotein of HTLV-1, HTLV-2, HTLV-3, HIV-1, HIV-2) of retrovirus, threadiness The fusion protein of viral fusion protein (for example, glycoprotein of EBOV, ZEBOV, REBOV, SEBOV and MARV), Arenavirus (for example, the glycoprotein of LASV, LCMV and JUNV and stabilization signal peptide (SSP)) and coronavirus fusion protein (for example, The S protein of HCoV, HToV, SARS-CoV and MERS-CoV).Representative Group III fusion protein includes the fusion egg of rhabdovirus It is white (for example, hydrophobin (RABV), Australian Bat rabies Tobamovirus (ABLV), bovine epizootic fever virus (BEFV) and The glycoprotein (G) of vesicular stomatitis virus (VSV)) and herpesviral fusion protein (for example, human herpesvirus 1's type (HHV-1； Also referred to as herpes simplex virus type 1 (HSV-1)), HHV-2 (also referred to as HSV-2), HHV-3 (also referred to as varicellazoster virus (VZV)), HHV-4 (also referred to as Epstein-Barr virus (EBV)) and HHV-5 (also referred to as cytomegalovirus (CMV) glycoprotein (gB, gD, gH/L))。

Extracellular domain polypeptide may include overall length precursor extracellular domain polypeptide or part thereof or be made from it.Some In embodiment, extracellular domain polypeptide lacks endogenous signal peptides, the endogenous head point of extracellular domain, extracellular domain Endogenous stem portion, endogenous mucin spline structure domain, endogenous film proximal end perimeter (MPER) and endogenous fusogenic peptide In any one or more persons.Alternatively or additionally, thus it is possible to vary or the one or more of missing wild type or reference fusion protein Endogenous protein cleavage sites (for example, one or more furin cleavage sites) are so that extracellular domain polypeptide is more It is not subject to the proteolytic cleavage of protease (for example, leukoprotease such as furin).

Can by understand overall length enveloped virus precursor fusion protein present in various structures part and funtion part or The position of structural domain constructs extracellular domain polypeptide of the invention.The structure of the extracellular domain polypeptide embodiment of reference example It builds, the non-limitative example of this kind of precursor protein and their relevant domain is discussed below.

2.2.1 Flu-A HA

Exemplary Flu-A HA precursor has following amino acid sequence:

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKIC NNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWT GVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTV STRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQ NSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALE NQHTIDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELK SGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI[SEQ ID NO:52](GenPeptgbAEC23340.1).This sequence includes following structural domain/part:

SP=1-16

Extracellular domain=17-529

Furin cleavage site=345-346

FP=346-355

Area=356 HRA -390

Area=421 HRB -470

MPER=470-529

TM=530-553

C=534-556

Head zone=51-328,403-444,

Pedicle region=17-58,327-401,442-509.

The non-limitative example of Flu-A HA extracellular domain polypeptide includes:

Extracellular domain 1-529:

MKTIIAFSCILCLIFAQKLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRIC NSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWT GVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIV STKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSE CITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQ NSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALE NQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLK SGYKD[SEQ ID NO:53](GenPept gbAEC23340.1)。

Deduct the extracellular domain 18-529 of SP:

KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDA LLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVN SFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRP WVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNV NKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAA INQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFE RTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD[SEQ ID NO: 54](GenPept gbAEC23340.1)。

Deduct the extracellular domain 18-469 that SP deducts MPER:

KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDA LLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVN SFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRP WVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNV NKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAA INQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFE RTRR[SEQ ID NO:55](GenPept gbAEC23340.1)。

Extracellular domain 18-341,346-529 additional changes furin cleavage site:

KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDA LLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVN SFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRP WVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNV NKITYGACPRYVKQNTLKLATGMRNVPERRRKKRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQ AAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKL FERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD[SEQ ID NO: 56](GenPept gbAEC23340.1)。

The additional joint area of stem structural domain 1-58,327-401,442-509:

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELGFGQNTLKLA TGMRNVPEKQTRGIFGAIAGFIENGWEGMLDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGMLNRLIGSGGSGEL LVALLNQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIK GVELKSGYKD[SEQ ID NO:57](GenPept gbAEC23340.1)。

The additional joint area of head domain 1-18,51-328,403-444:

MKTIIALSYILCLVFAQKEVTNATELVQNSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKW DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHSKFK YPALNVTMPNNEEFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTI VKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQN GSGGSGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELL[SEQ ID NO:58](GenPeptgbAEC23340.1)。

2.2.2 influenza B HA

Representative influenza B HA precursor has following amino acid sequence

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKL CPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTET APGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYG DSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKV IKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLE GGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDL RADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFS LPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL[SEQ ID NO:59] (GenPept gbAFH57854.1]。

This sequence includes following structural domain/part:

SP=1-16

Extracellular domain=17-547

Furin cleavage site=361-362

FP=362-382

The area HRA=383-416

The area HRB=436-487

MPER=488-547

TM=548-573

C=574-584

Head zone=48-344,418-456

Pedicle region=17-47,345-417,457-547

The non-limitative example of influenza B HA extracellular domain polypeptide includes:

Extracellular domain 1-547:

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKL CPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTET APGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYG DSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKV IKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLE GGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDL RADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFS LPTFDSLNITAASLNDDGLDNHT[SEQ ID NO:60](GenPept gbAFH57854.1)。

Deduct the extracellular domain 17-547 of SP:

RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGR PKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPN VANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTT HYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHE KYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSH GAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLS NEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLND DGLDNHT[SEQ ID NO:61](GenPept gbAFH57854.1)。

Deduct the extracellular domain 17-487 that SP deducts MPER:

RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGR PKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPN VANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTT HYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHE KYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSH GAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLS NEGIINSEDEHLLALERKLKKML[SEQ ID NO:62](GenPept gbAFH57854.1)。

The extracellular domain 17-355 of the furin cleavage site of the deduction additional change of SP, 362-547:

RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGR PKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPN VANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTT HYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHE KYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPARRRKKRAGFFGAIAGFLEGGWEGMIAGWHGYTS HGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLL SNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLN DDGLDNHT[SEQ ID NO:63](GenPept gbAFH57854.1)。

The additional joint area of stem structural domain 1-47,345-417,457-547:

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLGSGLANGTKYRPPAKLLKERG FFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSGSGGSGIELAVLLSNEGIINS EDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHT [SEQ ID NO:64](GenPept gbAFH57854.1)。

The additional joint area of head domain 1-17,48-344,418-456:

MKAIIVLLMVVTSNADRTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSI LHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWV IPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTE DEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTG EHAKAIGNCPIWVKTPLKGSGGSGELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQ[SEQ ID NO:65] (GenPept gbAFH57854.1)。

2.2.3RSV F

Non-limiting RSF F precursor has following amino acid sequence:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN IMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN[SEQ ID NO:66](GenPept gbAHL84194.1)。

This sequence includes following structural domain/part:

SP=1-23

Extracellular domain=24-524

Furin cleavage site=109-110,136-137

FP=137-163

The area HRA=164-196

The area HRB=488-524

TM=525-548

C=549-574

D25 interaction domain=61-97,193-240

The non-limitative example of RSV F extracellular domain polypeptide includes:

Extracellular domain 1-524:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN [SEQ ID NO:67](GenPept gbAHL84194.1)。

The extracellular domain (1-520) of RSV F:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK[SEQ ID NO:146]。

Deduct the extracellular domain 24-524 of SP:

SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVT ELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKI KSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGV TTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTS PLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKG EPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN[SEQ ID NO:68](GenPept gbAHL84194.1)。

Deduct the extracellular domain 24-524 of the furin cleavage site of the additional change of SP:

SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVT ELQLLMQSTQATNNNANNELPRFMNYTLNNAKKTNVTLSNNNNNNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKI KSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGV TTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTS PLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKG EPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN[SEQ ID NO:69](GenPept gbAHL84194.1)。

The additional joint area of D25 interaction domain 61-97,193-240:

LSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMGGLDLKNYIDKQLLPIVNKQSCSISNIETVI EFQQKNNRLLEITREFSVN[SEQ ID NO:70](GenPept gbAHL84194.1)。

2.2.4hMPV F

Schematic hMPV F precursor has following amino acid sequence

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIK TELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKT NEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLD LMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKK GNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISM VALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDP VKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSTMILVSVFIIIKKTKKPTGAP PELSGVTNNGFIPHN[SEQ ID NO:71](GenPept gbAAN52913.1)。

This sequence includes following structural domain/part:

SP=1-19

Extracellular domain=1-490

Furin cleavage site=102-103

FP=103-125

The area HRA=126-169

The area HRB=456-490

TM=491-514

C=515-539

The non-limitative example of hMPV F extracellular domain polypeptide includes:

Extracellular domain 1-490:

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIK TELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKT NEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLD LMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKK GNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISM VALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDP VKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:72](GenPeptgbAAN52913.1)。

Deduct the extracellular domain 20-490 of SP:

KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSAD QLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVR ELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAG QIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAG STVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESI ENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:73](GenPept gbAAN52913.1)。

Deduct the extracellular domain 20-490 of the furin cleavage site of the additional change of SP:

KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSAD QLAREEQIENPNQSNFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVR ELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAG QIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAG STVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESI ENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:74](GenPept gbAAN52913.1)。

2.2.5PIV F

Exemplary PIV F precursor has following amino acid sequence:

MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQI KQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDI EKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGS LQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKV DSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDI VPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNS VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTTIIIVLIMIIILFIINVTIIIIAVKYYRIQKR NRVDQNDKPYVLTNK[SEQ ID NO:75](GenPept gbAAB21447.1)。

This sequence includes following structural domain/part:

SP=1-19

Extracellular domain=1-493

Furin cleavage site=109-110

FP=110-135

The area HRA=136-168

The area HRB=458-493

TM=494-516

C=517-536

The non-limitative example of PIV F extracellular domain polypeptide includes:

Extracellular domain 1-493:

MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQI KQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDI EKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGS LQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKV DSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDI VPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNS VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:76](GenPeptgbAAB21447.1)。

Deduct the extracellular domain 20-493 of SP:

IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLR LQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQS SIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNI TEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSH IMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTT CTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDL EESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:77](GenPept gbAAB21447.1)。

Deduct the extracellular domain 20-493 of the furin cleavage site of the additional change of SP:

IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLR LQKDVIVSNQESNENTDPNTKNFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQS SIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNI TEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSH IMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTT CTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDL EESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:78](GenPept gbAAB21447.1)。

2.2.6MeV F

Representative MeV F precursor has following amino acid sequence:

MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNC TRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNS QAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPS LRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIV HRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVS GSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDL GPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTCIVYILIAVCLGGLIGIPALICCCRGRCNKK GEQVGMSRPGLKPDLTGTSKSYVRSL[SEQ ID NO:79](GenPept dbjBAB60865.1)。

This sequence includes following structural domain/part:

SP=1-24

Extracellular domain=1-493

Furin cleavage site=112-113

FP=113-137

The area HRA=138-171

The area HRB=454-493

TM=494-517

C=518-550

The non-limitative example of MeV F extracellular domain polypeptide includes:

Extracellular domain 1-493:

MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNC TRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNS QAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPS LRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIV HRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVS GSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDL GPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST[SEQ ID NO:80](GenPeptdbjBAB60865.1)。

Deduct the extracellular domain 25-493 of SP:

IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALN AMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAG QEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINK VLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVA TQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCK CYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLED AKELLESSDQILRSMKGLSST[SEQ ID NO:81](GenPept dbjBAB60865.1)

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALN AMTQNIRPVQSVASSNNHKNFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAG QEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINK VLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVA TQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCK CYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLED AKELLESSDQILRSMKGLSST[SEQ ID NO:82](GenPept dbjBAB60865.1)。

2.2.7HeV F

Non-limiting HeV F precursor has following amino acid sequence:

MATQEVRLKCLLCGIIVLVLSLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNV SKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQEL LPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSH VPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPP VYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPSLISMLSMIILYVLSIAALCIGLITFISFVIVEKKRGN YSRLDDRQVRPVSNGDLYYIGT[SEQ ID NO:83](GenPept NP_047111.2)。

This sequence includes following structural domain/part:

SP=1-20

Extracellular domain=1-487

Furin cleavage site=109-110

FP=110-135

The area HRA=136-169

The area HRB=456-587

TM=488-518

C=519-546

The non-limitative example of HeV F extracellular domain polypeptide includes:

Extracellular domain 1-487:

MATQEVRLKCLLCGIIVLVLSLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNV SKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQEL LPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSH VPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPP VYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS[SEQ ID NO:84](GenPept NP_047111.2)。

Deduct the extracellular domain 21-487 of SP:

SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILS PIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQET AEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYE TLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFV LIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTC QCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQ QSKDYIKEAQKILDTVNPS[SEQ ID NO:85](GenPept NP_047111.2)。

Deduct the extracellular domain 21-487 of the furin cleavage site of the additional change of SP:

SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILS PIKGAIELYNNNTHDLVGDVNLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQET AEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYE TLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFV LIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTC QCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQ QSKDYIKEAQKILDTVNPS[SEQ ID NO:86](GenPept NP_047111.2)。

2.2.8NiV F

Representative NiV F precursor has following amino acid sequence:

MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNM SQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQEL LPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSH VPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPP VFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNT YSRLEDRRVRPTSSGDLYYIGT[SEQ ID NO:87](GenPept NP 112026)。

This sequence includes following structural domain/part:

SP=1-20

Extracellular domain=1-487

Furin cleavage site=109-110

FP=110-135

The area HRA=136-169

The area HRB=456-487

TM=488-518

C=519-546

The non-limitative example of NiV F extracellular domain polypeptide includes:

Extracellular domain 1-487:

MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNM SQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQEL LPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSH VPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPP VFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS[SEQ ID NO:88](GenPept NP 112026)。

Deduct the extracellular domain of SP:

SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILT PIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQET AEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYE TLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFI LVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTC QCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQ QSKDYIKEAQRLLDTVNPS[SEQ ID NO:89](GenPept NP 112026)。

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILT PIKGALEIYKNNTHDLVGDVNLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQET AEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYE TLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFI LVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTC QCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQ QSKDYIKEAQRLLDTVNPS[SEQ ID NO:90](GenPept NP 112026)。

2.2.9HIV GP160

Schematic HIV GP160 precursor has following amino acid sequence:

MRVKGTRKNYWWRWGTMLLGMLMICSAAEQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWAT HACVPTDPNPQEVVLENVTENFNMWKNDMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTR NTTTANISRWEMEGEIKNCSFNVTTSIRNKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPI HYCTPAGFALLKCNDKKFNGTGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNET VVINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDP EIVMHSFNCGGEFFYCDTTQLFNSAWFSNNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITC SSNITGLLLTRDGGNNVTNESEIFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMF LGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGI WGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWN WFSISNWLWYIKIFIMIVAGLVGLRIFFAVLSIVNRVRQGYSPLSFQTHLPAQRGPDRPGGIEEEGGERDNGRSIR LVDGFLALIWDDLRSLCLFSYHRLRDLLLLVKRVVELLGHRGWEILKYWWNLLQYWSQELKNSAVSLFNAIAIAVA EGTDRVIEGIQRIGRGFLHIPRRIRQGLERALL[SEQ ID NO:91](GenPept dbjBAF31430.1)。

This sequence includes following structural domain/part:

SP=1-28

Extracellular domain=1-688

Furin cleavage site=508-509

FP=509-538

The area HRA=539-587

The area HRB=631-667

MPER=668-688

TM=689-711

C=712-861

GP41=509-861

GP120=1-508

The non-limitative example of HIV GP160 extracellular domain polypeptide includes:

Extracellular domain 1-688:

MRVKGTRKNYWWRWGTMLLGMLMICSAAEQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWAT HACVPTDPNPQEVVLENVTENFNMWKNDMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTR NTTTANISRWEMEGEIKNCSFNVTTSIRNKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPI HYCTPAGFALLKCNDKKFNGTGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNET VVINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDP EIVMHSFNCGGEFFYCDTTQLFNSAWFSNNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITC SSNITGLLLTRDGGNNVTNESEIFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMF LGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGI WGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWN WFSISNWLWYIK[SEQ ID NO:92](GenPept dbjBAF31430.1)。

Deduct the extracellular domain of SP:

EQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKND MVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIR NKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVS TVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATG DIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFS NNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGG GNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSG IVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIW NNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:93] (GenPept dbjBAF31430.1)。

Deduct the extracellular domain that SP deducts MPER:

EQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKND MVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIR NKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVS TVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATG DIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFS NNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGG GNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSG IVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIW NNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQ[SEQ ID NO:94](GenPept dbjBAF31430.1)。

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

EQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKND MVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIR NKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVS TVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATG DIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFS NNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGG GNMKDNWRSELYKYKVVKIEPLGVAPTNANNNVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSG IVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIW NNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:95] (GenPept dbjBAF31430.1)。

GP41 extracellular domain 509-688:

VVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVW GIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIE ESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:96](GenPept dbjBAF31430.1)。

2.2.10EBOV GP

Representative EBOV GP precursor has following amino acid sequence:

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGL NLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGT GPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKN LTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATIS TSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPAT TTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEG AAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGT CHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF [SEQ ID NO:97](GenPept NP_066246.1。

This sequence includes following structural domain/part:

SP=1-27

Extracellular domain=1-650

Furin cleavage site=501-502

Histone cleavage sites=191-192,201-202

FP=511-556

The area HRA=557-593

The area HRB=600-635

MPER=636-650

TM=651-669

C=670-676

Mucin spline structure domain=312-461

The non-limitative example of EBOV GP extracellular domain polypeptide includes:

Extracellular domain 1-650:

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGL NLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGT GPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKN LTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATIS TSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPAT TTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEG AAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGT CHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQ[SEQ ID NO:98](GenPept NP_ 066246.1)

Deduct the extracellular domain of SP:

QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPK VVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIY RGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESR FTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSP ARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDIS EATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESAS SGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQ DGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII HDFVDKTLPDQGDNDNWWTGWRQ[SEQ ID NO:99](GenPept NP_066246.1)。

Deduct the extracellular domain that SP deducts MPER:

QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPK VVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIY RGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESR FTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSP ARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDIS EATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESAS SGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQ DGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII HDFVDKTL[SEQ ID NO:100](GenPept NP_066246.1)。

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPK VVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIY RGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESR FTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSP ARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDIS EATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESAS SGKLGLITNTIAGVAGLITGGNNTNNEAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQ DGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII HDFVDKTL[SEQ ID NO:101](GenPept NP_066246.1)。

Deduct the extracellular domain that SP deducts mucin spline structure domain:

QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPK VVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIY RGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESR FTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVGGNNTHHQDTGE ESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGL MHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKI DQIIHDFVDKTLPDQGDNDNWWTGWRQ[SEQ ID NO:102](GenPept NP_066246.1)。

Deduct the extracellular domain in mucin spline structure domain:

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGL NLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGT GPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKN LTRKIRSEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAA EGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDW TKNITDKIDQIIHDFVDKTL[SEQ ID NO:154]

2.2.11MARV GP

Non-limiting MARV GP precursor has following amino acid sequence:

MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASK RWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFF LYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNS TKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPP TPSPQPSTPQQGGNNTNHSQDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITE NEQTSAPSITTLPPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFL DGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQED DLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGG TCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIF TKYIG[SEQ ID NO:103](GenPept YP_001531156.1)。

This sequence includes following structural domain/part:

SP=1-19

Extracellular domain=1-650

Furin cleavage site=434-435

FP=526-549

The area HRA=582-598

The area HRB=611-627

MPER=628-650

TM=651-669

C=670-681

Mucin spline structure domain=244-425

The non-limitative example of MARV GP extracellular domain polypeptide includes:

Extracellular domain 1-650:

MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASK RWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFF LYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNS TKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPP TPSPQPSTPQQGGNNTNHSQDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITE NEQTSAPSITTLPPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFL DGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQED DLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGG TCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG[SEQ ID NO:104](GenPept YP_ 001531156.1)。

Deduct the extracellular domain 20-650 of SP:

PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGE EAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEG NIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTAR PEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHS QDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPT TAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTI FDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGL YTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKN ISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG[SEQ ID NO:105](GenPept YP_001531156.1)。

Deduct the extracellular domain 20-627 that SP deducts MPER:

PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGE EAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEG NIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTAR PEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHS QDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPT TAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTI FDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGL YTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKN ISEQIDQI[SEQ ID NO:106](GenPept YP_001531156.1)。

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGE EAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEG NIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTAR PEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHS QDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPT TAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFNNNNSILWREGDMFPFLDGLINAPIDFDPVPNTKTI FDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGL YTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKN ISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG[SEQ ID NO:107](GenPept YP_001531156.1)。

Deduct the extracellular domain that SP deducts mucin spline structure domain:

PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGE EAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEG NIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTAR PEIKLGGAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLS YFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKS LELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWW TSDWG[SEQ ID NO:108](GenPept YP_001531156.1)。

2.2.12SARS-CoV S

Schematic SARS-CoV S precursor has following amino acid sequence:

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTG FHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPM GTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIF KLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEID KGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSAT KLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKL RPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQC VNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQD VNCTNVSAAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVA YTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGI AAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINA RDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQ IANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLIT GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERN FTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKG ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIA IVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT[SEQ ID NO:109](GenPeptgbAAR86788.1)。

This sequence includes following structural domain/part:

SP=1-13

Extracellular domain=1-1199

Popularity road trypsin like proteases cleavage site=667-668

FP=770-788

The area HRA=892-1013

The area HRB=1145-1187

MPER=1188-1199

TM=1200-1216

C=1217-1255

The non-limitative example of SARS-CoV S extracellular domain polypeptide includes:

Extracellular domain 1-1199:

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTG FHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPM GTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIF KLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEID KGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSAT KLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKL RPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQC VNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQD VNCTNVSAAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVA YTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGI AAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINA RDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQ IANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLIT GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERN FTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKG ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVW[SEQ ID NO:110](GenPept gbAAR86788.1)。

Deduct the extracellular domain of SP:

SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVI PFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRA ILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVP SGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYAD SFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFS PDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGV LTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTNVSAAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAY SNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFA QVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLT VLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQ ESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKA YFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKGELDKYFKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVW[SEQ ID NO:111](GenPeptgbAAR86788.1)。

Deduct the extracellular domain that SP deducts MPER:

SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVI PFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRA ILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVP SGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYAD SFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFS PDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGV LTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTNVSAAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAY SNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFA QVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLT VLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQ ESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKA YFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKGELDKYFKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK[SEQ ID NO:112](GenPeptgbAAR86788.1)。

2.2.13MERS-CoV S

Exemplary MERS-CoV S precursor has following amino acid sequence:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNIT ITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIY PAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYN RNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSII PHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSY PLSMKSDLGVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYS PCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL GNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGS VACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRS VPGEMRLASIAFNHPIQVDQFNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFC SKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPG YMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRL NGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQ RLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPN GLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQ VTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLK ELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH[SEQ ID NO:113](GenPept gbAHX00711.1)。

This sequence includes following structural domain/part:

SP=1-21

Extracellular domain=1-1301

Furin cleavage site=751-752,887-888

FP=888-891,951-980

The area HRA=984-1105

The area HRB=1248-1291

MPER=1292-1301

TM=1302-1318

C=1319-1353

The non-limitative example of MERS-CoV S extracellular domain polypeptide includes:

Extracellular domain 1-1301:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNIT ITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIY PAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYN RNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSII PHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSY PLSMKSDLGVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYS PCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL GNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGS VACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRS VPGEMRLASIAFNHPIQVDQFNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFC SKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPG YMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRL NGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQ RLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPN GLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQ VTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLK ELGNYTYYNKWPWYIWL[SEQ ID NO:114](GenPept gbAHX00711.1)。

Deduct the extracellular domain 22-1301 of SP:

GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFF NHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMY TYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYK LQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTP PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLGVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQL SPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSML KRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQF NSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNL FASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLIC AQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKF NQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTT LNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAY GLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGI DFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWL[SEQ ID NO:115](GenPept gbAHX00711.1)。

Deduct the extracellular domain 22-1291 that SP deducts MPER:

GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFF NHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMY TYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYK LQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTP PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLGVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQL SPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSML KRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQF NSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNL FASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLIC AQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKF NQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTT LNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAY GLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGI DFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTY[SEQ ID NO:116] (GenPept gbAHX00711.1)。

Deduct the extracellular domain of the furin cleavage site of the additional change of SP:

GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFF NHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMY TYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYK LQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTP PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLGVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQL SPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSML KRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPNSVNSVPGEMRLASIAFNHPIQVDQF NSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNL FASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSNSANSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLIC AQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKF NQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTT LNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAY GLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGI DFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWL[SEQ ID NO:117](GenPept gbAHX00711.1)。

2.2.14VSV G

Exemplary VSV G precursor has following amino acid sequence:

MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAI QADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTG FRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEREL WDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSK NPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLG[SEQ ID NO:118] (GenPept gbADX53329.1)。

This sequence includes following structural domain/part:

SP=1-17

Extracellular domain=1-462

MPER=421-462

TM=462-483

C=484-510

The non-limitative example of VSV G extracellular domain polypeptide includes:

Extracellular domain 1-462:

MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAI QADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTG FRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEREL WDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSK NPIELVEGWFSSWK[SEQ ID NO:119](GenPept gbADX53329.1)。

Deduct the extracellular domain of SP:

FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCD FRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVD SQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKM QYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPI SPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGV LRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK[SEQ ID NO:120](gbADX53329.1)。

Deduct the extracellular domain that SP deducts MPER:

FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCD FRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVD SQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKM QYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPI SPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGV LRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVF[SEQ ID NO:121](GenPept gbADX53329.1)。

2.2.15RABV GP

Exemplary RABV GP precursor has following amino acid sequence:

MIPQTLLFVPLLVFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVG YISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTV KTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRAS KGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELV KKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHP HVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSG VDLGLPSWGKYVLMSVGTLIALMLMILLTTCCRKANGAESIQHRLGETGRKVSVTSQNGRVISSWESYKSGGETKL [SEQ ID NO:122](GenPept gbAFM52658.1)。

This sequence includes following structural domain/part:

SP=1-20

Extracellular domain=1-458

TM=459-478

C=479-524

The non-limitative example of RABV GP extracellular domain polypeptide includes:

Extracellular domain 1-458:

MIPQTLLFVPLLVFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVG YISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTV KTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRAS KGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELV KKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHP HVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSG VDLGLPSWGK[SEQ ID NO:123](GenPept gbAFM52658.1)。

Deduct the extracellular domain of SP:

FPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAE TYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVSDLDPY DKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKG ACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVS FRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLI PEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSGVDLGLPSWGK[SEQ ID NO: 124](GenPept gbAFM52658.1)。

HSV1 Gb

Exemplary HSV1 Gb precursor has following amino acid sequence:

MRQGAPARGCRWFVVWALLGLTLGVLVASAAPSSPGTPGVAAATQAANGGPATPAPPALGAAPTGDPK PKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAESTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTE GIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLET TAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVY MSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEV DEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGF LIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRV AIAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRP LVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNIT MLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAAMFAGLGAFFEGMGDLGRAVGKV VMGIVGGVVSAVSGVSSFMSNPFGALAVGLLVLAGLAAAFFAFRYVMRLQSNPMKALYPLTTKELKNPTNPDASGE GEEGGDFDEAKLAEAREMIRYMALVSAMEHTEHKAKKKGTSALLSAKVTDMVMRKRRNTNYTQVPNKDGDADEDDL [SEQ ID NO:125](GenPept gbAAF04615.1)。

This sequence includes following structural domain/part:

SP=1-24

Extracellular domain=1-774

TM=775-795

C=796-904

The non-limitative example of HSV1 Gb extracellular domain polypeptide includes:

Extracellular domain 1-774:

MRQGAPARGCRWFVVWALLGLTLGVLVASAAPSSPGTPGVAAATQAANGGPATPAPPALGAAPTGDPK PKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAESTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTE GIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLET TAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVY MSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEV DEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGF LIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRV AIAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRP LVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNIT MLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAAMFAGLGAFFEGMGDLGRAVGKV VMGIVGGVVSAVSGVSSFMSNP[SEQ ID NO:126](GenPept gbAAF04615.1)。

Deduct the extracellular domain 25-775 of SP:

VLVASAAPSSPGTPGVAAATQAANGGPATPAPPALGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAA GHATLREHLRDIKAESTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDV TVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANAATRTS RGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTSYAADRFK QVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFT TNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGFLIAYQPLLSNTLAELYVREHLREQ SRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHELTLWNEARKLNPN AIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELR LTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLL DYTEVQRRNQLHDLRFADIDTVIHADANAAMFAGLGAFFEGMGDLGRAVGKVVMGIVGGVVSAVSGVSSFMSNP [SEQ ID NO:127](GenPept gbAAF04615.1)。

Enveloped virus fusion can be naturally present in for generating the extracellular domain polypeptide sequence of chimeric polyeptides of the present invention Active site of protein and/or it can have relative to natural fusion protein sequence it is one or more (for example, 1,2,3,4,5,6, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30) single ammonia Base acid mutation (insertion, missing or displacement).For example, as it is known that mutation F protein shears sequence with the furin for eliminating them, because And prevent processing intracellular.In specific embodiments, extracellular domain polypeptide lack any one in SP, TM and C-structure domain or More persons and optionally relative to natural fusion protein sequence contain it is one or more (for example, 1,2,3,4,5,6,7,8,9, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30) single amino acid is prominent Become (insertion, missing or displacement).

Any purpose enveloped virus fusion protein amino acid sequence can be used in the present invention, such as SEQ ID NO:2 to 127 Amino acid sequence, or the sequence with SEQ ID NO:2 to 127 with identity or similarity.Generally, it will be with SEQ ID NO: 2 to 127 have at least 75% identity or similarity, for example, with SEQ ID NO:2 to 127 have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity or similarity.

2.3 representative chimeric polyeptides constructs

The non-limitative example of chimeric polyeptides of the present invention is described below:

2.3.1 SSM of the influenza A virus HA extracellular domain-based on HIV GP160

MKTIIAFSCILCLIFAQKLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRIC NSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWT GVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIV STKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSE CITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQ NSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALE NQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLK SGYKDGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQN QQEKNEQELLE[SEQ ID NO:128]。

2.3.2 influenza A virus HA stem structural domain-SSM based on HIV GP160

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELGFGQNTLKLA TGMRNVPEKQTRGIFGAIAGFIENGWEGMLDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGMLNRLIGSGGSGEL LVALLNQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIK GVELKSGYKDGGRSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSL IEESQNQQEKNEQELLE[SEQ ID NO:129]。

2.3.3 SSM of the influenza A virus H5 extracellular domain-based on HIV GP160

MGWSCIILFLVATATGVHSEDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVK PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSS HEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISV GTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILRPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNA KCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWY GYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAEL LVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEIS GVKLESIGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQ NQPAKDEQELLE[SEQ ID NO:130]。

2.3.4 SSM of the influenza B HA extracellular domain-based on HIV GP160

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKL CPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTET APGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYG DSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKV IKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLE GGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDL RADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFS LPTFDSLNITAASLNDDGLDNHTGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDR EINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:131]。

2.3.5RSV SSM of the F extracellular domain-based on HIV GP160

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN GGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKN EQELLE[SEQ ID NO:132]。

2.3.6RSV F (1-520)-SSM based on HIV GP160:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSGIV QQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQPAKDEQELLE [SEQ ID NO:147]。

2.3.7RSV F (the 1-520)-DScav mutation-SSM based on HIV GP160:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNK CNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETV IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVD TVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSGIV QQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQPAKDEQELLE [SEQ ID NO:150]。

2.3.8hMPV SSM of the F extracellular domain-based on HIV GP160

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIK TELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKT NEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLD LMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKK GNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISM VALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDP VKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQA RILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:133]。

2.3.9PIV SSM of the F extracellular domain-based on HIV GP160

MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQI KQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDI EKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGS LQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKV DSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDI VPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNS VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTTGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIK QLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:134]。

2.3.10MeV SSM of the F extracellular domain-based on HIV GP160

MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNC TRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNS QAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPS LRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIV HRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVS GSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDL GPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIK QLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:135]。

2.3.11HeV SSM of the F extracellular domain-based on HIV GP160

MATQEVRLKCLLCGIIVLVLSLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNV SKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQEL LPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSH VPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPP VYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPSGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI LAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:136]。

2.3.12NiV SSM of the F extracellular domain-based on HIV GP160

MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNM SQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNI NKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQD PVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQEL LPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSH VPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPP VFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI LAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:137]。

2.3.13HIV SSM of the GP160 extracellular domain-based on RSV F

MRVMGIERNYPCWWTWGIMILGMIIICNTAENLWVTVYYGVPIWKDANTTLFCASDAKAYDTEVHNVW ATHACVPTDPSPQELKMENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVQLTPLCVTLDCSYNITNNITNSITN SSVNMREEIKNCSFNMTTELRDKNRKVYSLFYKLDVVQINNGNNSSNLYRLINCNTSALTQACPKVTFEPIPIHYC APAGYAILKCNDKEFNGTGLCKNVSTVQYTHGIRPVVSTQLLLNGSLAEGKVMIRSENITNNVKNIIVQLNESVTI NCTRPNNNTRRSVRIGPGQTFYATGDIIGDIRQAHCNVSGSQWNKTLHQVVEQLRKYWNNNTIIFNSSSGGDLEIT THSFNCAGEFFYCNTSGLFNSTWVNGTTSSMSNGTITLPCRIKQIINMWQRVGQAMYAPPIQGVIKCESNITGLIL TRDGGVNSSDSETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKARRRVVEREKRAVTLGAVFIGFLGTAGSTM GAVSITLTVQARKLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICPT NVPWNSSWSNKSLDEIWENMTWLQWDKEISNYTIKIYELIEESQIQQERNEKDLLELDKWASLWNWFDISKWLWYI KGGGVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKGGGGHHHHGGGFDASISQVNEKINQSLAFIRKSDELLHNV [SEQ ID NO:138]。

2.3.14 the EBOV GP extracellular domain (1-311,462-650)-for reducing mucin spline structure domain is based on HIV The SSM of GP160

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGL NLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGT GPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKN LTRKIRSEELSFTVVGGNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTT QDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQR WGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQGGSGIVQQQNNLLRAIEAQQHL LQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:139]。

2.3.15 the MARV GP extracellular domain-SSM based on HIV GP160 in mucin spline structure domain is reduced

MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASK RWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFF LYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNS TKNQTCAPSKIPPPLPTARPEIKLGGAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSS GASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIK NQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQ IKKDEQKEGTGWGLGGKWWTSDWGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDR EINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:140]。

2.3.16SARS-CoV SSM of the S extracellular domain-based on HIV GP160

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTG FHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPM GTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIF KLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEID KGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSAT KLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKL RPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQC VNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQD VNCTNVSAAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVA YTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGI AAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINA RDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQ IANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLIT GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERN FTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKG ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWGGSGIVQQQ NNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:141]。

2.3.17MERS-CoV SSM of the S extracellular domain-based on HIV GP160

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNIT ITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIY PAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYN RNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSII PHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSY PLSMKSDLGVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYS PCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL GNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGS VACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRS VPGEMRLASIAFNHPIQVDQFNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYICNGFQKCEQLLREYGQFC SKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPG YMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRL NGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQ RLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPN GLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQ VTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLK ELGNYTYYNKWPWYIWLGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:142]。

2.3.18VSV SSM of the G extracellular domain-based on HIV GP160

MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAI QADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTG FRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEREL WDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSK NPIELVEGWFSSWKGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLI HSLIEESQNQQEKNEQELLE[SEQ ID NO:143]。

2.3.19RABV SSM of the GP extracellular domain-based on HIV GP160

MIPQTLLFVPLLVFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVG YISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTV KTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRAS KGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELV KKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHP HVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSG VDLGLPSWGKGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLI EESQNQQEKNEQELLE[SEQ ID NO:144]。

2.3.20HSV1 SSM of the Gb extracellular domain-based on HIV GP160

MRQGAPARGRRWFVVWALLGLTLGVLVVSAAPSSPGTPGVAAATQAANGGPATPAPPAPGAPPTGDPK PKKNKKPKPPKPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTE GIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLET TAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVY MSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEV DEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGF LIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRV AIAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRP LVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNIT MLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAAMFAGLGAFFEGMGDLGRAVGKV VMGIVGGVVSAVSGVSSFMSNPGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDRE INNYTSLIHSLIEESQNQQEKNEQELLE[SEQ ID NO:145]。

2.4 use structure stabilization part as general oligomerization domain

Structure stabilization part is in addition to the conformation side after stablizing extracellular domain polypeptide confrontation of the invention and being rearranged into fusion Except the practicability in face, structure stabilization part is also used as general oligomerization domain (UOD) to can be used for making any heterologous purpose Molecule oligomerization is melted into oligomer, especially tripolymer.In a particular embodiment, UOD melts in heterologous protein molecule upstream or downstream It closes to form chimeric polyeptides.Generally, UOD is merged in the downstream of heterologous protein molecule.Implement with extracellular domain as described herein Scheme is the same, and the complementary heptapeptide repetitive sequence of UOD is formed each other under conditions of (for example, in aqueous solution) being suitable for their associations Conjunction results in antiparallel double helix beam and is formed, the antiparallel double helix beam trimerizing to form highly stable Six helix bundle, Thus allow chimeric polyeptides trimerizing to form trimeric polypeptide complex.

Heterologous protein molecule can be natural polypeptides or non-native polypeptide.In certain embodiments, heterologous polypeptide be or Include treatment polypeptide.A variety of treatments known in the art can be used for treating a variety of diseases with polypeptide (including ligand and receptor). The known a variety of examples of target and the treatment indication of polypeptide are shown in table 8.

Table 8

UOD of the invention can be used to generate trimerized soluble receptor, for example including TNF receptor superfamily member, Ig Superfamily member, cytokine receptor superfamily member, chemokine receptors superfamily member, integrins group member, life Growth factor receptor body family, hormone receptor, Opioid Receptors, other neuropeptide receptors, ion channel etc., including CD1a-e, CD2(LFA-2)、CD2R、CD3γ、CD3δ、CD3ε、CD4-7、CD8a、CD8b、CD9、CD10 CD11a、CD11b、CD11c、 CDwl2、CD13、CD14、CD15、CD15s、CD15u、CD16a(FcγRIIIA)、CD16b(FcγRIIIB)、CDw17、CD18 (integrin β 2), CD19-28, CD29 (integrin β 1), CD30, CD31 (PE-CAM-1), CD32 (Fc γ RII), CD33 (Siglec-3)、CD34-41、CD42a-d、CD43、CD44、CD44R、CD45、CD45RA、CD45RB、CD45RO、DC47、 CD47R、CD48、CD49a-f(VLA-1-6)、CD50(ICAM-3)、CD51、CD52、CD53、CD54(ICAM-1)、CD55、 CD56(N-CAM)、CD57、CD58(LFA-3)、CD59、CD60a-c、CD61、CD62E、CD62L、CD62P、CD63、CD64、 CD65、CD65s、CD66a-f、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD79a、 CD79b、CD80、CD81、CD82、DC83、CDw84、CD85、CD86-CD91、CDw92、CD93、CD94-CD99、CD99R、 CD100-CD106、CD107a、CD107b、CD108-CD112、CDw113、CD114(G-CSFR)、CD115(M-CSFR)、 CD116, CD117, CD118, CDw119, CD120a, CD120b, CD121a (IL-1R, I type), CDw121b (IL-1R, II type), CD122(IL-2Rβ)、CDw123(IL-3R)、CD124(IL-4R)、CDw125(IL-5R)、CD126(IL-6R)、CD127(IL- 7R)、CDw128、CDw128b(IL-8Rβ、CD129(IL-9R)、CD130(IL-6Rβ)、CDw131、CD132、CD133、CD134 (Ox-40)、CD135-CD139、CD140a(PDGFRα)、CD140b(PDGFRβ)、CD141-CD144、CDw145、CD146、 CD147、CD148、CD15、CD151、CD152(CTLA-4)、CD153(CD30L)、CD154(CD40L)、CD155、CD156a- c、CD157、CD158a、CD158b、CD159a、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、 CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、 CD175、CD175s、CD176、CD178(FasL)、CD179a、CD179b、CD180、CD181(CXCR1)、CD182(CXCR2)、 CD183(CXCR3)、CD184(CXCR4)、CD185(CXCR5)、CDw186(CXCR6)、CD191(CCR-1)、CD192 (CCR2)、CD193(CCR3)、CD194(CCR4)、CD195(CCR5)、CD196(CCR6)、CD197(CCR7)、CDw198 (CCR8), CDwl99 (CCR9), CD200 (Ox-2), CD201, CD202b, CD203c, CD204 (macrophage scavenger R), CD207 (langhans' cells differential protein (Langerin)), CD208 (DC-LAMP), CD209 (DC-SIGN), CDw210 (IL- 10R)、CD212(IL-12-Rβ1)、CD213a1(IL-13-Rα1)、CD213a2(IL-13-Rα2)、CDw217(IL-17-R)、 CDw218a (IL-18R α), CDw218b (IL-18R β), CD220 (insulin-R), CD221 (IGF-1R), CD222 (IGF-II R), CD223-234, CD235a (glycophorin A), CD235ab (glycophorin A/B), CD235b (glycophorin B), CD236 (glycophorin C/D), CD236R (glycophorin C), CD238, CD239, CD240CE, CD240D, CD241- CD249、CD252(Ox40L)、CD254(RANKL)、CD256(APRIL)、CD257(BAFF)、CD258(LIGHT)、CD261 (TRAIL-R1)、CD262(TRAIL-R2)、CD263(DcR1)、CD264(DcR2)、CD256(RANK)、CD266(TWEAK- R)、CD267(TACI)、CD268(BAFFR)、CD269(BCMA)、CD271(NGFR)、CD272(BTLA)、CD273(PD-L2)、 CD274(PD-L1)、CD275(B7-H2)、CD276(B7-H3)、CD277、CD278(ICOS)、CD279(PD1)、CD280、 CD281(TLR1)、CD282(TLR2)、CD283(TLR3)、CD284(TLR4)、CD289(TLR9)、CD292、CDw293、 CD294, CD295 (leptin R), CD296, CD297, CD298 (3 subunit of Na+/K+ATP enzyme β), CD299 (L-SIGN), CD300a、CD300c、CD300e、CD301-CD307、CD309(VEGF-R2)、CD312、CD314-322、CD324、CDw325、 CD326、CDw327、CDw328、CDw329、CD331-CD337、CDw338、CD339、B7-H4、Xedar、CCR10、CCR11、 CX3CR1, chemokine-like receptor -1 (ChemR23), complement receptors, DARC, IL-11R, IL-12R, IL-13R, IL-15R, IL-20R, IL-21R, IL-22R, IL-23R, IL-27R, IL-28R, IL-31R, XCR1, CX3CR1, chemokine binding protein 2 (D6), interferon receptors, leucocyte correlation Ig sample receptor family, leukocytic immunity globulin sample receptor family include LILRC1 With LILRC2, leukotriene receptor, LAMP, handle protein like proteins 1-4, IgSF8, immunoglobulin-like transcript family LT1-6, EDAR, interstitial derivative factor (SDF), thymic stroma lymphocytic series receptor, EPO Receipter, promote blood platelet Generate plain receptor, EGF-R ELISA, fibroblast growth factor acceptor FGF1-4, hepatocyte growth factor receptor (HGF-R), epaCAM, insulin-like growth factor receptor IGF1-R and IGF2-R, fibronectin, fibronectin leucine are abundant Transmembrane protein FLRT1-3, Her2,3 and 4, CRELD1 and 2,8D6A, lipoprotein receptor (LDL-R), c-type agglutinin family at Member such as CLEC-1, CLEC-2, CLEC4D, 4F and Dectin 1 and 2, layilin, growth hormone receptor, prolactin releasing hormone Receptor (PRRP), corticotropin-releasing hormone receptor (CRHR), Follicle Stimulating Hormone Receptors (FSHR), gonadotropic hormone are released Put hormone receptor (GNRHR), thyrotrophin-releasing hormone receptor (TRHR), somatostatin receptor SSTR1-SSTR5, pitressin Receptor 1A, 1B, 2, ocytocin receptor, metakentrin/human chorionic gonadotropin receptor (LHCGR), thyrotropin receptor, Atrial natriuretic peptide receptor NPR1-3, acetylcholinergic receptor (AChR), calcitonin receptor (CT), cholecystokinin receptor CCKAR and CCKBR, vip receptor VPAC1 and 2, delta-opioid receptor, κ-Opioid Receptors, μ-opioid Receptor, sigma-receptor σ 1 and σ, Cannabined receptor R1 and 2, angiotensin receptor AT1-4, bradykinin receptor V1 and 2, tachykinin by Body 1 (TACR1), Calcitonin receptor-like receptor (CRLR), galanin receptors R1-3, GPCR neuropeptide receptor Neuropeptide B/W R1 and 2, neuropeptide FF receptor R1 and R2, neuropeptide S receptor R1, neuropeptide Y receptor Y1-5, Neurotensin receptor, I type and II activation Plain receptor, Activin receptor-like kinases (Alk-1 and Alk-7), β proteoglycans, BMP and activin film mating type inhibit albumen (BAMBI), cripto, Trk receptor TrkA, TrkB, TrkC, axl receptor family, LTK receptor family, TIE-1, TIE-2, Ryk, Neuropilin 1, Eph receptor EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, melanocortin receptor MC-3 and MC-4, AMICA, The sex-limited T cell mating type molecule of CXADR, corticoliberim-binding protein, I type, MHCI, MHCII, Ampoterin induced gene and ORF (AMIGO), APJ, asialoglycoprotein-receptors 1 and 2 (ASGPR), brain specificity blood Pipe generate inhibit albumen 3 (BAI-3), basal cell adhesion molecule/Luther glycophorin (BCAM/Lu), cadherin, CDCP1, cystic fibrosis transmembrane conductance regulator MRP-7, cartilage aggregation albumen, lung surface active element, tight junction protein, (epithelial cell sticks point by ANTHXR2, collagen, complement receptors, contactin 1-6, cubulin, endoglycan, EpCAM Son), endothelial protein C receptor (EPCR), Eph receptor, GLP-1 receptor GLP-1R and 2R, glutamate receptor, grape Saccharide transporter, Glycine Receptors, phosphatidyl alcohol proteoglycans, G- albumen Farnesoid X receptor in pairs, G- G-protein linked receptor 15, KLOTHO family member, leptin receptor, LIMPII, LINGO, NOGO, 1 (LYVE- of lymphatic endothelial hyalomitome saccharide acceptor 1), marrow sample inhibition c-type agglutinin receptor CLEC12A, regenerated protein (neogenin), nephrosis albumen (nephrin), NETO-1, NETO-2, nmda receptor, opioid associativity cell adhesion molecule, osteoclast inhibition agglutinin are related Albumen, tumour inhibitor receptor, osteoclast-associated receptor, bone activin (osteoactivin), thrombin receptor, flatfoot albumen, Preceding cell is swollen die receptor (porimin), potassium channel, Pref-1, stem cell factor receptor, brain signal albumen (semaphorin), SPARC, scavenger receptor-A 1, stabilin, syndecan, T cell receptor, TCAM-1, T cell cytokine receptor TCCR, blood platelet response protein, TIM1-6, toll sample receptor, the triggering receptor and TREM sample expressed on myeloid cell (TREM) Albumen, TROP-2 or its is any quasi- like object or the like.

In addition, UOD of the invention can be used to make any one ligand trimerizing of aforementioned receptor, the receptor for example including TNF superfamily member, cell factor superfamily member, growth factor, chemotactic factor (CF) superfamily member, angiogenic factors, Promote antiapoptotic factors, integrin, hormone and other soluble factors etc., including RANK-L, lymphotoxin (LT)-α, LT- β, LT- α1β2、zLIGHT、BTLA、TL1A、FasL、TWEAK、CD30L、4-1BB-L(CD137L)、CD27L、Ox40L(CD134L)、 GITRL、CD40L(CD154)、APRIL(CD256)、BAFF、EDA1、IL-1α、IL-1β、IL-1RA、IL-2、IL-3、IL-4、 IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、IL-16、IL-17A、IL- 17F、IL-17A/F、IL-18、IL-1g、IL-20、IL-21、IL-22、IL-23、IL-24、IL-25、IL-26、IL-27、IL- 28、IL-29、IL-30、IL-31、IL-32、IL-33、IL-34、IL-35、IFN-γ、IFN-α、IFN-β、TNF-α、TNF-β、 G-CMF, GM-CSF, TGF-β 1,2 and 3, TGF- α, cardiotrophin-1, LIF ELISA (LIF), β cytokine (betacellulin), amphiregulin, thymic stroma lymphocytic series (TSLP), flt-3, CXCL1-16, CCL1-3, CCL3L1, CCL4-CCL8, CCL9/10, CCL11-28, XCL1, XCL2, CX3CL1, HMG-B1, heat shock protein, Chemerin, sozin, macrophage migration inhibition factor (MIF), oncostatinM, limitin, vascular endothelial growth factor VEGF A-D and PIGF, lens epithelium derivative growth factor, hematopoietin, thrombopoietin, blood platelet spread out Raw growth factor, epidermal growth factor, fibroblast growth factor FGF1-14 and 16-23, hepatoma derivative growth factor, Hepassocin, hepatocyte growth factor, blood platelet-derivative endothelium growth factor (PD-ECGF), insulin-like growth factor IGF1 and IGF2, igf binding protein (IGFBP1-6), GASPS (the relevant haemocyanin of Growth and Differentiation Factors), connective tissue Growth factor, epigen, epiregulin (epiregulin), developmental character artery and neural crest epidermal growth factor (DANCE), Glia maturation factor-β, insulin, growth hormone, angiogenin, Ang-1-4, angiopoietin-like protein 1-4,5 β 1 of beta 2 integrin alpha V β 3, α V β 5 and α, hematopoietin, thrombopoietin, prolactin releasing hormone, promote kidney Upper gland cortin releasing hormone (CRH), gonadotropin-releasing hormone, thyrotrophin-releasing hormone, growth hormone release inhibiting hormone, pressurization Element, oxytocins, sandopart, carbetocin, metakentrin (LH) and human chorionic gonadtropin, thyroid swash Element, ANP, BNP, CNP, calcitonin, CCK a, CCK B, vasoactive intestinal peptide 1 and 2, enkephalins, dynorphin, beta-endorphin, Coffee, 4-PPBP, [1] SA 4503, dimethylbenzene guanidine (Ditolylguanidine), siramesine angiotensins, kallidins, Bradykinin, tachykinin, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, calcitonin, galanin, neurotensin, neuropeptide Y 1-5, neuropeptide S, neuropeptide FF, nerve Peptide B/W, neurotrophic factor derived from brain BDNF, NT-3, NT-4/5, activin A, AB, B and C, inhibin, Miao's Le Shi pipe inhibit Hormone (MIH), bone morphogenic protein BMP-2 2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15, growth and differentiation factor GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, myostatin/GDF8, GDF9, GDF10, GDF11, GDF15, nerve growth factor (NGF), neurotrophic factor derived from brain (BDNF), neurotrophic factor -3 (NT-3) match with neurotrophic factor -4 (NT-4), artemin, persephin, neural order albumen, GDNF, collectin, liver Protein ligands EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNB1, EFNB2, EFNB3, adiponectin, alpha2-macroglobulin, Agrecan, agouti chromoprotein related protein (AgRP), α-melanocyte-stimulatinghormone, albumin, ameloblast albumen (ameloblastin), plasminogen, angiostatin, Apolipoprotein A1, AII, B, B100, E, amyloid protein, Autophagy proteins (autophagin), TGF-β induction protein I g H3), Biglycan, the derivative chemotactic of leukocyte cell Factor LECT2, C reactive protein, complement component, notochord generate element, notochord generates plain sample albumen, collectins, Clusterin Sample albumen 1, cortisol, the van willebrandt factor, cell chalone (cytostatin), Endostatin, Endoreppellin, ephrins ligand, myosin (fetuin), ficolin, glucagon, particle cytolysin, Gremlin, HGF activator inhibit albumen HAI-1 and 2, kallikrein (kallilcrein), laminin, leptin, Lipocalin, mannan-binding lectin (MBL), nickel line albumen (meteorin), MFG-E8, macrophage galactolipin N- second Acyl group-galactosamine specific agglutination plain (MGL), Midkine, actin, nestin (nestin), osteoblast are special The factor 2, osteopontin (osteopontin), bone knit element (osteocrin), bone adhesion protein (osteoadherin), positive five Polyprotein, persephin, placenta growth factor, relaxain, Resistin protein and Resistin protein sample molecule, stem cell factor, This calcium element (stanniocalcins), VE- statins, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, tenascin, vitronectin, tissue factor, tissue because In sub- approach restrainer and being identified in mankind's secretion group as what is listed in secreted protein database > 7000 kinds of protein Any other protein (Chen Y et al., 2005.Nucleic Acids Res 33Database Issue:D169-173) Or its is any quasi- like object or the like.

In addition, UOD of the invention can be used to make enzyme such as angiotensin converting enzyme (ACE), matrix metalloproteinase, ADAM metalloproteinases (ADAMTS 1,4,5,13), aminopeptidase, the site β APP with blood platelet response protein I type motif Nickase (BACE-1 and -2), chymotrypsin, kallikrein, reelin, serine protease inhibitor (serpin) or Its is any quasi- like object or the like trimerizing.

In addition, UOD of the invention can be used to make chemotherapeutic and toxin such as bacterial origin, fungal source, plant source or animal sources Small molecule toxins or enzyme activity toxin or its segment trimerizing, the effect of to increase target compound for therapeutic purposes, example Such as calicheamycin, Pseudomonas exotoxin, diphtheria toxin, ricin, Saponaria officinalis toxin protein, apoptosis induction peptide or its is any Analog.

In other embodiments, UOD of the invention can also be used to the antigen of fusion Theratope, such as colorectal cancer resists Former A33, alpha-fetoprotein, mucin 1 (MUC1), CDCP1, carcinomebryonic antigen cell adhesion molecule, Her-2,3 and 4, mesothelin, CDCP1, NETO-1, NETO-2, syndecan, LewisY, CA-125, melanoma associated antigen (MAGE), tyrosinase, on Skin tumour antigen (ETA) etc., and fusion envelope antigen or fungal antigen, for treating communicable disease.

2.5 methods for preparing chimeric polyeptides construct

Chimeric polyeptides of the invention can be prepared by chemical synthesis or recombinant means.In general, by suitable host The recombinant precursor that encoding chimera polypeptide is expressed in cell, prepares chimeric polyeptides, but any suitable method can be used.It closes Suitable host cell is for example including insect cell (for example, Aedes aegypti (Aedes aegypti), autographa california (Autographa californica), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), Spodopterafrugiperda (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni)), mammalian cell (example Such as, people, non-human primates, horse, ox, sheep, dog, cat and rodent (for example, hamster), avian cells (for example, chicken, duck and goose), Bacterium is (for example, Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis) and streptococcus Species (Streptococcus spp.)), yeast cells (for example, saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), Candida maltosa (Candida maltosa), multiple-shaped nuohan inferior yeast (Hansenula polymorphs), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Ji Shi Pichia pastoris (Pichia guillerimondii), pichia pastoris yeast (Pichia pastoris), schizosaccharomyces pombe (Schizosaccharomyces pombe) and Yarrowialipolytica Asia yeast (Yarrowia lipolytica)), tetrahymena (Tetrahymena) cell is (for example, tetrahymena thermophila (Tetrahymena Thermophile)) or combinations thereof.It is well known that many suitable insect cells and mammalian cell.Suitable insect is thin Born of the same parents (are derived from parent for example including Sf9 cell, Sf21 cell, Tn5 cell, Schneider S2 cell and High Five cell The clone and separate strain (Invitrogen) of cabbage looper BTI-TN-5B1-4 cell line).Suitable mammalian cell for example wraps Include Chinese hamster ovary (CHO) cell, human embryonic kidney cells (HEK293 cell, the general Adenovirus Type 5 DNA conversion with shearing Cross), NIH-3T3 cell, 293-T cell, Vero cell, HeLa cell, PERC.6 cell (ECACC deposit number 96022940), Hep G2 cell, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), rhesus macaque tire pneumonocyte (ATCC CL-160), Madin-Darby ox kidney (" MDBK ") cell, Madin-Darby dog kidney (" MDCK ") cell (for example, MDCK(NBL2),ATCC CCL34；Or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cell, such as BHK21-F, HKCC cell etc..Suitable avian cells for example including chicken embryo stem cell (for example,Cell), chicken embryo fibroblasts, Chicken embryo reproduction cell, duck cell in such as Vaccine 27:4975-4982 (2009) and WO2005/042728 (for example, retouch AGE1.CR the and AGE1.CR.pIX cell line (ProBioGen) stated), EB66 cell etc..

Suitable insect cell expression system, such as rhabdovirus system, be known to the skilled in the art and In Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987) Description.For baculoviral/insertion cell expression system material and method in a kit form especially from Invitrogen, San Diego Calif is available commercial.Avian cells expression system is also known to the skilled in the art and for example U.S. Patent number 5,340,740；5,656,479；5,830,510；6,114,168；With 6,500,668；European patent number EP 0787180B；European Patent Application No. EP03291813.8；WO 03/043415；It is described in WO 03/076601.Similarly, Bacterial cell and mammalian cell expression system are also known in the art and for example in Yeast Genetic It is described in Engineering (Barr et al. writes, 1989) Butterworths, London.

Conventional method can be used and prepare the recombinant precursor for encoding chimeric polyeptides of the present invention in suitable carrier.In elder brother A variety of suitable carriers that recombinant protein is expressed in worm cell or mammalian cell are it is well known that and conventional.It is suitable to carry Body can contain numerous components, the component include but is not limited to it is following one or more: replication orgin；Selectable marker gene； One or more expression control elements, as transcriptional control element (for example, promoter, enhancer, terminator), and/or it is a kind of or A variety of translation signals；(for example, mammal source or from heterologous mammal or non-mammalian species) is used for Targeted to the signal sequence or leader sequence of secretory pathway in selected host cell.For example, being closed to be expressed in insect cell Suitable rhabdovirus expression vector can be used to generate recombinant baculovirus particle such as pFastBac (Invitrogen).By bar Shape virion expands and is used to infected insect cell, to express recombinant protein.In order to express in mammalian cells, use The carrier of construct expression will be driven in purpose mammalian host cell (for example, Chinese hamster ovary cell).

Any suitable method purifying chimeric polyeptides can be used.It is well known that the suitable side of purifying target protein Method, including precipitation and a plurality of types of chromatographies, such as hydrophobic interaction chromatograph, ion-exchange chromatography, affinity chromatography, chelate chromatography And size exclusion chromatography.The two or more of these or other appropriate method can be used, generate suitable purification schemes.Root It may include the purification part or " label " for promoting purifying according to needs, chimeric polyeptides, as described in the part 2.1.2.It can lead to Chelate chromatography or affinity chromatography are crossed, such as advantageously purifies this kind of tagged polypeptide from conditioned medium.

Chimeric polyeptides may include additional sequence.For example, for expression purpose, it can be by the natural of purpose heterologous polypeptide Leader peptide (for example, native leader peptide of enveloped virus fusion protein) replaces with a different leader peptide.

3. for the endogenous nucleic acid construct for generating chimeric polyeptides

The present invention also conceives in host organism, suitably vertebrate, preferably endogenous generation in mammal (such as people) The nucleic acid construct of chimeric polyeptides.Nucleic acid construct can be the outer carrier/replicon (for example, plasmid) of self-replacation type chromosome Or it is integrated into the carrier of host genome.In a particular embodiment, nucleic acid construct is viral vectors.Exemplary poisonous carrier It is carried including retroviral vector, slow virus carrier, poxvirus vector, vaccinia virus vector, adenovirus vector, adeno-associated virus Body, herpesvirus vector, jaundice poisonous carrier and alphavirus vectors.Viral vectors can be living, attenuation, copy condition type or multiple Deficiency processed, and usually non-pathogenic (defective), the viral vectors for having replication capacity.

By way of example, when viral vectors is vaccinia virus vector, the multicore of chimeric polyeptides of the present invention will can be encoded Thuja acid is inserted into the essential site of vaccinia virus viral vector gene group.This kind of essential site is for example in Perkus et al. (1986.Virology 152:285)；Hruby et al. (1983.Proc.Natl.Acad.Sci.USA 80:3411)；Weir etc. Description in people (1983.J.Virol.46:530).It include but is not limited to P7.5 with the matching used suitable promoter of vaccinia virus (for example, with reference to Cochran et al. 1985.J.Virol.54:30)；P11 (for example, with reference to Bertholet, et al., 1985.Proc.Natl.Acad.Sci.USA 82:2096)；With CAE-1 (for example, with reference to Patel et al., 1988.Proc.Natl.Acad.Sci.USA 85:9431).The highly attenuated strain of vaccinia virus is more acceptable in human body simultaneously And Lister, NYVAC including being lacked containing specific gene group (for example, with reference to Guerra et al., 2006.J.Virol.80: 985-998)；Tartaglia et al., 1992.AIDS Research and Human Retroviruses 8:1445-1447) Or MVA (for example, with reference to Gheradi et al., 2005.J.Gen.Virol.86:2925-2936)；Mayr et al., 1975.Infection 3:6-14).It sees also Hu et al. (2001.J.Virol.75:10300-10308), describes sub- bar sample Virus is used for the purposes of cancer therapy as carrier)；U.S. Patent number 5,698,530 and 6,998,252.Such as see also the U.S. The patent No. 5,443,964.See also U.S. Patent number 7,247,615 and 7,368,116.

In certain embodiments, adenovirus vector can be used for expressing purpose chimeric polyeptides.Baculovirus transfer vector can be with It can come from any source, any subgroup, any hypotype, mixing hypotype or any serotype based on adenovirus thereon.For example, Adenovirus may belong to subgroup A (for example, serotype 12,18 and 31), subgroup B (for example, serotype 3,7,11,14,16,21, 34,35 and 50), subgroup C (for example, serotype 1,2,5 and 6), subgroup D (for example, Serotype8,9,10,13,15,17,19,20, 22-30,32,33,36-39 and 42-48), it is subgroup E (for example, serotype 4), subgroup F (for example, serotype 40 and 41), unfiled Sero-group (for example, serotype 49 and 51) or any other adenoviral serotype.From American type culture collection (ATCC, Manassas, Va.) can get adenoviral serotype 1 to 51.Non- group C adenovirus and even non-human adenovirus can be with For preparing replication-defective adenoviral vector.Such as in U.S. Patent number 5,801,030,5,837,511 and 5,849,561 and Non- group C adenovirus vector is disclosed in international patent application WO 97/12986 and WO 98/53087, generates non-group C adenovirus load The method of body and the method for using non-group C adenovirus vector.Any adenovirus, even chimeric adenoviral, can be used as adenopathy The viral genome source of poisonous carrier uses.For example, adenovirus hominis can be used as the viral base of replication-defective adenoviral vector Because group source uses.It can be in Molin et al. (1998.J.Virol.72:8358-8361), Narumi et al. (1998.Am J.Respir.Cell Mol.Biol.19:936-941), Mercier et al. (2004.Proc.Natl.Acad.Sci.USA 101:6188-6193), US publication 20150093831,20140248305,20120283318,20100008889, 20090175897 and 20090088398 and U.S. Patent number 6,143,290；6,596,535；6,855,317；6,936,257； 7,125,717；7,378,087；The other examples of adenovirus vector are found in 7,550,296.

Viral vectors can also be based on adeno-associated virus (AAV).About the description of the carrier based on AAV, for example, with reference to beauty State's patent No. 8,679,837,8,637,255,8,409,842,7,803,622 and 7,790,449 and US publication 20150065562,20140155469,20140037585,20130096182,20120100606 and 20070036757.AAV Carrier is also possible to (sc) AAV carrier of self-complementary, these carriers are for example in 2007/01110724 He of U.S. Patent Publication 2004/0029106 and U.S. Patent number 7,465,583 and 7,186,699 in describe.

Viral vectors based on herpes simplex virus (HSV) applies also for endogenous generation chimeric polyeptides of the invention.It is many The hsv vector of replication defective contains the missing for removing one or more immediate early genes to prevent duplication.Herpesvirus vector The advantages of be it into the latency stage that can cause DNA long-term expression ability, and its up to 25kb exogenous DNA can be accommodated Big viral DNA genome.About the description of the carrier based on HSV, for example, with reference to U.S. Patent number 5,837,532,5,846, 782,5,849,572 and 5,804,413 and international patent application WO 91/02788, WO 96/04394, WO 98/15637 and WO 99/06583。

Retroviral vector may include based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), those of ecotropic retrovirus, monkey immunodeficiency virus (SW), human immunodeficiency virus (HIV) and combinations thereof Carrier is (for example, with reference to Buchscher et al., 1992.J.Virol.66:2731-2739；Johann et al., 1992.J.Virol.66:1635-1640；Sommerfelt et al., 1990.Virology 176:58-59；Wilson et al., 1989.J.Virol.63:2374-2378；Miller et al., 1991.J.Virol.65:2220-2224；Miller et al., 1990.Mol.Cell Biol.10:4239；Kolberg,1992.NIH Res.4:43；Cornetta et al., 1991.Hum.Gene Ther.2:215)。

In a particular embodiment, retroviral vector is slow virus carrier.Such as the skilled person will understand that, virus carries Body, such as slow virus carrier are often referred to the vector particles comprising viral vector gene group.For example, slow virus carrier particle can To include lentiviral vector genome group.For slow virus carrier, vector gene group can be derived from a variety of suitable, available The carrier based on lentiviral gene group any one, including it is identified for Human Gene Therapy application those of carrier (example Such as, referring to Pfeifer et al., 2001.Annu.Rev.Genomics Hum.Genet.2:177-211).Suitable slow virus carries Body genome includes based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious poor Those of blood virus, monkey immunodeficiency virus (SIV) He Meidi/Wei Sina virus.The desirable feature of slow virus is it Can infect fissility and overstepping one's bounds fragility cell, but target cell it is unnecessary be fissility cell or it is unnecessary be stimulated with Division.In general, genome and envelope glycoprotein be by based on different virus, thus resulting vector particles pseudotyping.It closes It is incorporated to the security features of viral vectors with needing.Security features include that as described in more detail herein self is devitalized LTR and integrate defect.In certain embodiments, integrating defect can be assigned by the element of vector gene group, it is also possible to spread out The element of packaging system is born from (for example, the nonfunctional integration that can not be the part of vector gene group but supply in a manner of trans- Zymoprotein).Exemplary carrier contain packaging signal (psi), Rev- response element (RRE), donor splicing site, acceptor splicing site, optionally Ground center polypurine region (central poly-purine tract；) and WPRE element cPPT.In certain exemplary embodiment party In case, viral vector gene group includes the sequence from lentiviral gene group (such as HIV-1 genome or SIV genome).Virus Genome construct may include the sequence from slow virus 5' and 3'LTR, and especially may include from slow virus 5'LTR R sequence and U5 sequence and from the inactivation type of slow virus or self devitalized 3'LTR.LTR sequence, which can be to come to be derived from, appoints The LTR sequence of any slow virus of what species.For example, they can be the LTR sequence from HIV, SIV, FIV or BIV.One As, LTR sequence is HIV LTR sequence.

Vector gene group may include inactivation type or self devitalized 3'LTR (for example, with reference to Zufferey et al., 1998.J.Virol.72:9873；Miyoshi et al., 1998.J.Virol.72:8150).Self devitalized carrier usually has Enhancer sequence and promoter sequence missing from 3' long terminal repeats (LTR), the missing is during vector integration It copies in 5'LTR.In one case, the U3 element of 3'LTR contains its enhancer sequence, TATA frame, the site Spl and NF- κ The missing in the site B.As self it is devitalized 3'LTR's as a result, enter and reverse transcription after the provirus that generates will be comprising inactivation Type 5'LTR.By reducing the mobile risk of vector gene group and reducing influence of the LTR to neighbouring cellular promoters, improve safety Property.Self devitalized 3'LTR can be constructed by any method known in the art.

It is optionally possible to the U3 sequence from slow virus 5'LTR be replaced with the promoter sequence in virus constructs, such as Heterologous promoter sequence.This can increase the titre of the virus recycled from package cell line.Also it may include enhancer sequence.It can So as to be used in any enhancers/promoters combination for increasing the expression of viral RNA genes group in package cell line.In an example In, it uses cmv enhancer/promoter sequence (for example, with reference to U.S. Patent number 5,385,839 and 5,168,062).

It in certain embodiments, is to minimize insertion mutation wind with defect is integrated by building slow virus carrier Danger.It can seek kinds of schemes and generate non-integrating vectors genome.These schemes require to dissolve into (all) such engineerings of mutation The integrase enzyme component of pol gene, so that it encodes the protein with inactive integrase.Vector gene group sheet can be modified Body, such as by being mutated or lacking one or two attachment site or make the proximal end 3'LTR- polypurine area by missing or modification Domain (PPT) nonfunctional, prevents from integrating.In addition, non-heredity scheme is available；These schemes include inhibiting one or more integration The pharmacological agents of enzyme function.These schemes do not have to be mutually exclusive, that is, can be every time using more than one in these schemes.For example, Integrase and attachment site can be with nonfunctional or attachment sites and the site PPT with nonfunctional or integrase and the site PPT It can be with nonfunctional with nonfunctional or all of which.

Such as US publication 20150224209,20150203870,20140335607,20140248306, Exemplary slow virus carrier is described in 20090148936 and 20080254008.

Viral vectors can also be based on Alphavirus.Alphavirus includes sindbis alphavirus (and Venezuelan equine encephalitis virus (VEEV)), Aura virus, Babanki virus, bar horse forest virus, Bebaru virus, Cabassou virus, chikungunya disease Poison, eastern equine encephalitis, Everglades are viral, Fort Morgan virus, Getah is viral, Highlands J is viral, Kyzylagach virus, Mayaro are viral, Me Tri virus, Middelburg is viral, Mosso das Pedras is viral, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, ross river virus, Salmon Pancreas Disease virus, Semliki forest virus (SFV), southern elephant seal virus, Tonate virus, Trocara virus, Una Virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus and Whataroa virus.In general, the genome of this viroid is compiled The non-structural protein (for example, replicon) that can be translated in the cytoplasm of host cell of code and structural proteins (for example, capsid with Coating).The virus transfer that ross river virus, sindbis alphavirus, SFV and VEEV have been used to develop delivering transgenosis carries Body.Pseudotyped viral can be formed by combining envelope glycoprotein and retroviral capsid.It can be in US publication 20150050243, the example of alphavirus vectors is found in 20090305344 and 20060177819.

Alternatively, viral vectors can be based on flavivirus.Flavivirus includes japanese encephalitis virus, dengue virus (for example, stepping on Remove from office hot -1, dengue fever -2, dengue fever -3, dengue fever -4), yellow fever virus, Murray Valley encephalitis virus, St. Louis encephalitis Virus, West Nile Virus, Kunjin virus, Rocio encephalitis viruses, Ilheus virus, tick-brone encephalitis virus, central European encephalitis disease Poison, Siberi encephalitis viruses, russian spring-summer encephalitis virus, Kyasanur disease virus, msk haemorrhagia fever virus, Louping-ill virus, Powass virus, Negishi virus, Absettarov virus, Hansalova virus, Apoi virus and Hypr Virus.Can US publication 20150231226,20150024003,20140271708,20140044684, 20130243812、20120294889、20120128713、20110135686、20110014229、20110003884、 20100297167, it looks in 20100184832,20060159704,20060088937,20030194801 and 20030044773 To the example of jaundice poisonous carrier.

4. chimeric polyeptides complex

Chimeric polyeptides of the invention can under suitable conditions self-assembly to form chimeric polyeptides complex.Therefore, this hair It is bright to further contemplate that a kind of method for generating chimeric polyeptides complex, the method comprise the steps that compound being suitable for chimeric polyeptides Combination chimeric polyeptides of the invention under conditions of (for example, in aqueous solution) body is formed, thus generate chimeric polyeptides complex, institute Chimeric polyeptides complex is stated to include three chimeric polyeptides and be characterized in that the curling that the respective structure forming part of chimeric polyeptides divides Helical structure is formed by Six helix bundle.The chimeric polyeptides being combined can be identical or not identical, thus is respectively formed same trimerization Body and heterotrimer.

In general, chimeric polyeptides self-assembly in buffered aqueous solution (for example, pH about 5 to about 9).As needed, it can be used Mild Denaturing e.g. by being included in urea, a small amount of organic solvent or heating be leniently denaturalized chimeric polyeptides, promotes again Folding and self-assembly.

Any suitable chimeric polyeptides preparation process may be incorporated in this method.For example, containing there is purpose chimeric polyeptides Conditioning cell culture medium can be used in this method.However, it is preferred in the method using the chimeric polyeptides of purifying.

Keep extracellular domain polypeptide oligomerization chimeric to be formed using structure stabilization part/general oligomerization domain In the specific embodiment of complex of polypeptides, the extracellular domain polypeptide moiety of complex, which is in, merges preceding conformation.Be not intended to by Any particular theory or operation mode constraint, it is believed that form is as described herein multiple before the fusion of extracellular domain polypeptide tripolymer It is stabilized in zoarium, is formed the reason is that heterologous structural stabilizes part induction complex and prevent extracellular domain polypeptide Interior section or structural domain (for example, the area HRA and the area HRB of I class extracellular domain polypeptide, or the C in Group III extracellular domain The central alpha-helix coiled coil and fusion ring of end regions) interaction.The interaction of this kind of interior section or structural domain is led Cause refolding at form after fusion.

5. screening technique

Present invention also contemplates that screening is preferably specifically bound with enveloped virus fusion protein and/or fusion protein complex Substance method.In a particular embodiment, to library of compounds screening, to merge extracellular domain more with envelope virus is contained The combination of the chimeric polyeptides of peptide or its complex.

Candidate substances cover numerous chemicals classifications, including for example small organic compound of small molecule and macromolecular such as peptide, polypeptide And polysaccharide.Candidate substances include interact required functional group, especially hydrogen bond with protein structural, and generally comprise to Lack amine, carbonyl, hydroxy or carboxy, desirably at least two functional chemical groups.Candidate compound may include through one The cyclic annular carbon structure or heterocycle structure or aromatics or more aromatic structures that a or multiple above-mentioned functional groups replace.Candidate substances there is also In following biomolecule, the biomolecule includes but is not limited to peptide, sugar, fatty acid, steroids, purine, pyrimidine, derivative Object, analogue or combinations thereof.Library of compounds may include in bacterial extract, fungal extract, plant extracts With the native compound of animal extracts form.Alternatively or additionally, library of compounds may include native compound or conjunction At the compound of generation.

It is known in the art for determining something whether in conjunction with target protein and/or something is to the affinity of target protein Method.It is, for example, possible to use multiple technologies detection and/or the combinations of quantitative material and target protein, and the technology is such as, but not limited to, Biological bilayer interferometry (BioLayer Interferometry, BLI), immunoblotting, Dot blot, surface etc. from Daughter resonance method (SPR), enzyme linked immunosorbent assay (ELISA) (ELISA),OrMeasurement Method is based on mass spectrographic method.

In some embodiments, it can be used known in the art any based on surface plasma body resonant vibration (SPR) Measuring method analyzes substance, wherein the measuring method is for characterizing substance and the chimeric polyeptides of the present invention containing extracellular domain polypeptide Or the kinetic parameter of composite bulk phase interaction.It can use any commercially available SPR instrument in method described herein, including but It is not limited to BIAcore instrument (Biacore AB；Uppsala, Sweden)；1Asys instrument (Affinity Sensors；Franklin, Mass.)；IBIS system (Windsor Scientific Limited；Berks, UK), SPR-CELLIA system (Nippon Laser and Electronics Lab；Hokkaido, Japan) and SPR Detector Spreeta (Texas Instruments；Dallas, Texas).For example, with reference to Mullett et al. (2000) Methods 22:77-91； Dong et al. (2002) Reviews in Mol Biotech 82:303-323；Fivash et al. (1998) Curr Opin Biotechnol 9:97-101；With Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

In some embodiments, BLI can be used to measure in substance and contain on Octet (ForteBio Inc.) Bio-molecular interaction between the chimeric polyeptides or complex of the present invention of extracellular domain polypeptide.BLI is by surveying in real time The thickness change of protein layer in biosensor tips is measured, the ligand being fixed in biosensor tips is perceived and (such as contains The chimeric polyeptides or complex of the present invention of extracellular domain polypeptide) and solution in analyte (such as test compound) between combine Marker free optical analysis technique.

In some embodiments, AlphaScreen (PerkinElmer) measuring method can be used to characterize for examination substance with The combination of chimeric polyeptides or complex of the present invention containing extracellular domain polypeptide.Initial ALPHA represents amplification type Shine short range homogeneous assay method.AlphaScreen is the short range measuring method based on pearl, by measurement because of donor bead and acceptor bead Between signal caused by energy transfer, perceive molecule (such as theme chimeric polyeptides or compound engaged with donor bead and acceptor bead Body and test compound) between combination.(for example, with reference to Eglen et al. (2008) Curr Chem Genomics 1:2- 10)。

In some embodiments,(PerkinElmer) measuring method can be used to characterize for trying object The combination of matter and chimeric polyeptides of the present invention or complex.AlphaLISA is improved from above-mentioned AlphaScreen measuring method, to include Acceptor bead containing europium, and alternatively playing a role as traditional ELISA measuring method.(for example, with reference to Eglen et al. (2008) Curr Chem Genomics 1:2-10.)。

Panimmunity determination techniques, including competitive and non-competitive immunoassays can be used.Term " immunoassays Method " covers many technologies, including but not limited to flow cytometry, FACS, enzyme immunoassay (EIA), as enzyme Multiple immunizations are surveyed Determine technology (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA) and particulate enzyme Immunoassay (MEIA) also comprises but is not limited to capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunoradiometry (IRMA), fluorescence polarization immunoassay (FPIA) and chemiluminescence assay (CL).If It needs, this kind of immunoassay can automate.Immunoassay can also be used in combination with the fluorescence of induced with laser.Liposome Immunoassay, such as flowing injection liposome immunization measuring method and Liposome Immunosensor, are also applied in the present invention.This Outside, scattering turbidimetry measuring method (nephelometry assay) is suitable for the method for the present invention, wherein for example protein/antibody is multiple The formation for closing object causes light scattering to increase, this is converted to the peak rate signal for changing with marker concentration and changing.

In some embodiments, it can be used and be related to differential scanning fluorescence method (DSF) and differential static light scattering (DSLS) thermal denaturation method measures the combination for trying substance and theme chimeric polyeptides or complex.

In some embodiments, the method based on mass spectrography can be used, e.g., but be not limited to and mass spectrography (AS-MS) The affinity back-and-forth method of Platform Alliance analyzes the combination for trying substance and chimeric polyeptides of the invention or complex.This is one The method of kind marker free, wherein protein and test compound are incubated, unbonded molecule is washed away and matched by identification The MS of body analyzes protein-ligand complexes, is followed by a solution composite steps.

In some embodiments, can for example using the protein of detectable label, as radio-labeled (for example,³²P、³⁵S、¹⁴C or³H), the chimeric polyeptides or complex or test compound of fluorescent marker (for example, FITC) or enzymatic labelling, by exempting from Epidemic disease measuring method is detected by chromatography, the quantitative combination for trying substance and theme chimeric polyeptides or complex.

In some embodiments, present inventive concept is in directly or indirectly measurement chimeric polyeptides or complex and for trying chemical combination Fluorescent polarization assay and fluorescence resonance energy transfer (FRET) measuring method are used when the degree to interact between object.

Above-mentioned whole embodiment is adapted to exploitation into high-throughput platform.

Compound can be further tested, in animal model to identify that those have the chemical combination of most strength vivo efficacy Object, for example, specifically binding with enveloped virus fusion protein or fusion protein complex and preferably stimulating or enhance treatment has With those of effect (for example, virus load is reduced, infection or associated symptom are reduced) compound.For example, by making chemical combination The successively modification, other routine programs used in molecule modeling and Rational drug design of object experience, these molecules can serve as into " lead compound " of one step exploitation drug.

6. antigen binding molecules

Chimeric polyeptides and complex of the present invention containing extracellular domain can be used for generating antigen binding molecules, preferably It is the protein (that is, " antigen-binding proteins ") with the interaction of enveloped virus fusion protein immunization.In a particular embodiment, Chimeric polyeptides and complex containing extracellular domain include at least one after the fusion of enveloped virus fusion protein in form Epitope before the fusion being not present, and therefore can be used for preparing and the meta-stable form of enveloped virus fusion protein or merge preceding shape The antigen binding molecules of the immune interaction of formula.

Those of ordinary skill in the art will be appreciated that the fully developed knowledge about antigen-binding proteins, for example, such as Abbas Et al., Cellular and Molecular Immunology, the 6th edition, W.B.Saunders Company (2010) or Murphey et al., Janeway's Immunobiology, the 8th edition, described in Garland Science (2011), the text The each piece offered completely is incorporated herein by reference by reference.

In some embodiments, the antigen-binding proteins with chimeric polyeptides of the present invention and the immune interaction of complex are Antibody.As defined described in part, antibody includes complete antibody and its antigen-binding fragment.Antibody may include complete antibody point Son (including with total length heavy chain and/or the polyclonal of light chain, monoclonal, chimeric, humanization or person form) includes its antigen Binding fragment.Antibody fragment includes F (ab')₂, Fab, Fab', Fv, Fc and Fd segment, and can be incorporated to single domain antibody, Single-chain antibody, big antibody (maxibody), miniantibody, intracellular antibody, Diabody, three body antibody, four body antibody, v-NAR With double-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23,9,1126- 1136).It further include antibody polypeptides, those antibody polypeptides as disclosed in U.S. Patent number 6,703,199, including fibronectin are more Peptide monomer antibody.Other antibody polypeptides are disclosed in U.S. Patent Publication 2005/0238646, they are single chain polypeptides.

Numerous preparations are known in the art for the method for the antibody of purpose antigen.It is, for example, possible to use the miscellaneous of routine Tumor method is handed over to generate the monoclonal antibody for being directed to chimeric polyeptides of the present invention and complex, the hybridoma method is often based on Initiative method (1975, " Continuous Cultures Of Fused Cells Secreting of Kohler, G. et al. Antibody Of Predefined Specificity, " Nature 256:495-497) or its modification.Generally, non- Monoclonal antibody is formed in human species (such as mouse).In general, mouse or rat are used for immunity inoculation, but other also can be used Animal.Immunogene (being in the present case chimeric polyeptides or complex of the invention) immunity inoculation with immunogenicity amount can be passed through Mouse generates antibody.Immunogene can time (e.g., once every two weeks or once a week) application repeatedly, or can at regular intervals It is applied in a manner of by vigor is maintained in animal.

In order to monitor antibody response, biological small sample (for example, blood) and testing needle can be obtained from animal to immunogene Antibody titer.Spleen and/or several big lymph nodes can be taken out and be dissociated into individual cells.If desired, application can be passed through Plate or hole of the cell suspension to antigen coat, screening splenocyte (after removing non-specific attached cell).Expression is to antigen The B cell of special film mating type immunoglobulin will not be leached in conjunction with plate and with the rest part of suspension and leave away. Resulting B cell or the splenocyte all dissociated then can be with myeloma cells (for example, X63-Ag8.653 and coming from Salk Institute, cell Distribution Center, San Diego, those of Calif.) fusion.Polyethylene glycol (PEG) can be used to make spleen Or lymphocyte is merged with myeloma cell to form hybridoma.Then in selective medium (for example, hypoxanthine, amino Pterin, thymidine medium, further referred to as " HAT culture medium ") in cultivate hybridoma.Then as obtained by limiting dilution assay cover plant Hybridoma, and for example screened using FACS (cell sorting methods of fluorescence-activation) or immunohistochemistry (IHC), analysis with The generation for the antibody that immunogens combine.Then in vitro (for example, in tissue culture flasks or hollow fiber reactor) Or the hybridoma of selected secrete monoclonal antibody is cultivated in vivo (for example, as the ascites in mouse).

Another as cell fusion object technology is alternative, the B cell that Epstein-Barr virus (EBV) immortalizes can be used to generate with Theme chimeric polyeptides or the monoclonal antibody of the immune interaction of complex.As needed, hybridoma is expanded and is subcloned, and And pass through conventional analysis program (for example, FACS, IHC, radiommunoassay, enzyme immunoassay (EIA), fluorescence immunoassay etc.) point Analyse the former activity of anti-immunity of supernatant.

Therefore, the present invention has been further contemplated that generation and enveloped virus fusion protein or fusion protein complex are immune mutual The method of the antigen binding molecules of effect, the method comprise the steps that (1) is fitted into as the above and elsewhere this paper is broadly described Complex of polypeptides or composition immunity inoculation animal, wherein the extracellular domain polypeptide of chimeric polyeptides complex corresponds to coating disease Malicious fusion protein；(2) from the B cell of animal separation and fusion protein or the immune interaction of fusion protein complex；(3) it produces The raw antigen binding molecules expressed by this B cell.Present invention also contemplates that by such methods generate antigen binding molecules with And its derivative.The cell that can generate antigen binding molecules of the present invention, including hybridoma are also covered, and is generated from those cells The method of antigen binding molecules.In a particular embodiment, the antigen binding molecules generated by the method for the present invention and cell are excellent Selection of land is neutrality antigen binding molecules.

It is contemplated that chimeric antibody and humanized antibody.In some embodiments, Humanized monoclonal antibodies include mouse The variable domains (or entirety or part of its antigen binding site) of antibody and the constant domain derived from human antibody.Alternatively Ground, the antigen binding site and the variable domains derived from human antibody that humanized antibody segment may include mouse monoclonal antibody Segment (lacks antigen binding site).Program for generating engineering monoclonal antibody is included in Riechmann et al., 1988,Nature 332:323；Liu et al. people, 1987, Proc.Nat.Acad.Sci.USA 84:3439；Larrick et al., 1989,Bio/Technology 7:934；With Winter et al., described in 1993, TIPS 14:139 those.In a reality It applies in scheme, chimeric antibody is the antibody of CDR transplanting.Such as in U.S. Patent number 5,869,619；5,225,539；5,821, 337；5,859,205；6,881,557；Padlan et al., 1995, FASEB J.9:133-39；Tamura et al., 2000, J.Immunol.164:1432-41；Zhang, W., et al., Molecular Immunology 42 (12): 1445-1451, 2005；Hwang W. et al., Methods 36 (1): 35-42,2005；Dall'Acqua W F, et al., Methods 36 (1): 43-60,2005；And Clark, M., Immunology Today 21 (8): discussing in 397-402,2000 makes antibody humanization Technology.

Antibody of the invention is also possible to human monoclonal antibodies.It can be by appointing known to those of ordinary skill in the art The technology of what number generates human monoclonal antibodies.Such methods include but is not limited to that Epstein-Barr virus (EBV) conversion human peripheral is thin Born of the same parents' (for example, containing bone-marrow-derived lymphocyte), ion vitro immunization inoculation human B cell, the spleen of transgenic mice of the fusion from immunity inoculation are thin Born of the same parents, the transgenic mice carry the human immunoglobulin gene of insertion, separated from human immunoglobulin(HIg) V area's phage library, Or as known in the art and other programs based on the disclosure.

The program that human monoclonal antibodies are generated in non-human animal is developed.For example, having prepared wherein The mouse of one or more endogenous immunoglobulin genes is inactivated by multiple means.Human immunoglobulin gene is drawn Enter mouse, to substitute the murine genes of inactivation.In this art, people's heavy chain and the introducing of the element of light chain gene seat are derived from The mouse species of embryonic stem cell line, the embryonic stem cell line contains endogenous heavy chain and the targeting of light chain gene seat destroys (referring also to Bruggemann et al., Curr.Opin.Biotechnol.8:455-58 (1997)).For example, human immunoglobulin(HIg) Transgenosis can be minigene construct or transgenosis seat on yeast artificial chromosome, and the latter is in mouse lymph sample tissue The rearrangement of B cell specific DNA and hypermutation occurs.

The antibody generated in animal is incorporated to by the human immunoglobulin(HIg) polypeptide chain for the people's inhereditary material coding for introducing animal.In In one embodiment, theme chimeric polyeptides or complex immunogen immune are inoculated with non-human animal, such as transgenic mice.

Described in hereafter document for generating transgenic animals and generating human antibody or part using transgenic animals The example of the technology of human antibody: U.S. Patent number 5,814,318,5,569,825 and 5,545,806；Davis et al., Production of human antibodies from transgenic mice, writes Antibody quoted from Lo Engineering:Methods and Protocols,Humana Press,NJ:191-200(2003)；Kellermann etc. People, 2002, Curr Opin Biotechnol.13:593-97；Russel et al., 2000, Infect Immun.68:1820- 26, Gallo et al., 2000, Eur J.Immun.30:534-40；Davis et al., 1999, Cancer Metastasis Rev.18:421-25；Green,1999,J Immunol Methods 231:11-23；Jakobovits,1998,Advanced Drug Delivery Reviews 31:33-42；Green et al., 1998, J Exp Med.188:483-95；Jakobovits A,1998,Exp.Opin.Invest.Drugs 7:607-14；Tsuda et al., 1997, Genomics 42:413-21； Mendez et al., 1997, Nat.Genet.15:146-56；Jakobovits,1994,Curr Biol.4:761-63； Arbones et al., 1994, Immunity 1:247-60；Green et al., 1994, Nat.Genet.7:13-21； Jakobovits et al., 1993, Nature 362:255-58；Jakobovits et al., 1993, Proc Natl Acad Sci USA 90:2551-55；Chen, J., M. et al. Int.Immunol.5 (1993): 647-656；Choi et al., 1993, Nature Genetics 4:117-23；Fishwild et al., 1996, Nature Biotech.14:845-51；Harding et al., 1995,Annals of the New York Academy of Sciences；Lonberg et al., 1994, Nature 368: 856-59；Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies, quoted from Handbook of Experimental Pharmacology 113:49-101；Lonberg et al., 1995, Int.Rev.Immunol.13:65-93；Neuberger,1996,Nature Biotech.14:826；Taylor et al., 1992,Nucleic Acids Research 20:6287-95；Taylor et al., 1994, Int.Immunol.6:579-91； Tomizuka et al., 1997, Nature Genetics 16:133-43；Tomizuka et al., 2000, Proc Natl Acad Sci USA 97:722-27；Tuaillon et al., 1993, Proc Natl Acad Sci USA 90:3720-24 and Tuaillon et al., 1994, J.Immunol.152:2912-20.；Lonberg et al., Nature 368:856,1994； Taylor et al., Int.Immunol.6:579,1994；U.S. Patent number 5,877,397；Bruggemann et al., 1997Curr.Opin.Biotechnol.8:455-58；Jakobovits et al., 1995.Ann.N.Y.Acad.Sci.764: 525-35.In addition, for example in U.S.05/0118643 and WO 05/694879, WO 98/24838, WO 00/76310 and the U.S. It describes and is related in the patent No. 7,064,244The scheme of (Abgenix is now Amgen, Inc.).

Present invention further contemplates that the segment of the anti-chimeric polyeptides/recombinant antibody of the present invention.This kind of segment can completely by Sequence derived from antibody forms or may include additional sequence.The example of antigen-binding fragment includes Fab, F (ab')₂, it is single-stranded Antibody, Diabody (diabodies), three body antibody (triabodies), four body antibody (tetrabodies) and structural domain are anti- Body.Lunde et al. provides other examples in 2002, Biochem.Soc.Trans.30:500-06.

Single-chain antibody can be formed in the following manner: by heavy-chain variable domains and light variable domains (area Fv) piece Section is connected through amino acid bridge (short peptide linkers), to generate single polypeptide chain.By more in two kinds of variable domains of coding Peptide (V_LAnd V_H) DNA between fusion coding peptide linker DNA, be prepared for this kind of scFv (scFv).Resulting polypeptide can be certainly Row inflection is folded can to form polymer (for example, dimer, tripolymer or four are poly- to form antigen-binding monomer or they Body), this depend on flexible joint between two variable domains length (Kortt et al., 1997, Prot.Eng.10:423； Kortt et al., 2001, Biomol.Eng.18:95-108).It include V by combination_LAnd V_HNot homopolypeptide, can be formed with not The polymer scFv (Kriangkum et al., 2001, Biomol.Eng.18:31-40) combined with epitope.To generate single-chain antibody The technology of exploitation is included in U.S. Patent number 4,946,778；Bird,1988,Science 242:423；Huston et al., 1988,Proc.Natl.Acad.Sci.USA 85:5879；Ward et al., 1989, Nature 334:544；De Graaf etc. People, described in 2002, Methods Mol.Biol.178:379-87 those.

It can also for example be hydrolyzed by the proteolytic of antibody, for example, pepsin or pawpaw according to conventional methods Protease digestion complete antibody obtains the antigen-binding fragment for being derived from antibody.It by way of example, can be by with pepsin The enzymatic cutting of antibody is carried out to provide referred to as F (ab')₂5S segment, generate antibody fragment.Sulfydryl can be used in this segment Reducing agent is further cut, to generate 3.5S Fab' monovalent fragment.It is optionally possible to which the blocking groups using sulfydryl are cut Reaction is cut, caused by the mercapto gene-splicing disulfide bond.Alternately, two are directly produced using the enzymatic cutting of papain A monovalent Fab fragment and Fc segment.These methods are for example by the United States Patent (USP) of Goldenb 4,331,647；Nisonoff et al., Arch.Biochem.Biophys.89:230,1960；Porter,Biochem.J.73:119,1959；Edelman et al. draws From Methods in Enzymology 1:422 (Academic Press 1967)；And Andrews, S.M. and Titus, J.A., quoted from Current Protocols in Immunology (Coligan J.E., et al. write), John Wiley& Sons, New York (2003), the 2.8.1-2.8.10 pages and the 2.10A.1-2.10A.5 pages description.Also other can be used The method of cutting antibody (such as separation heavy chain to form monovalent light-heavy chain segment (Fd), depth cutting segment) or other enzymes are changed Or genetic technique, as long as the antigen binding that the segment is identified by complete antibody.

Another form of antibody fragment is the peptide of one or more complementary determining regions (CDR) comprising antibody.It can lead to The polynucleotides for crossing building coding purpose CDR, obtain CDR.For example, by using the mRNA of antibody produced cell as template, Using polymerase chain reaction synthetic variable region, this kind of polynucleotides are prepared (for example, with reference to Larrick et al., Methods:A Companion to Methods in Enzymology 2:106,1991；Courtenay-Luck,"Genetic Manipulation of Monoclonal Antibodies, " quoted from Monoclonal Antibodies:Production, Engineering and Clinical Application, Ritter et al. (writing), (Cambridge of page 166 University Press 1995)；With Ward et al., " Genetic Manipulation and Expression of Antibodies, " quoted from Monoclonal Antibodies:Principles and Applications, Birch et al. (writing), page 137 (Wiley-Liss, Inc.1995)).Antibody fragment can also include at least the one of antibody as described herein A Variable domain.Thus, for example, it is that V region structural domain can be monomer and be can be at least equal to 10^-7M or smaller Affinity independently combine theme extracellular domain polypeptide or the V of its complex_LAnd V_HStructural domain.

Variable domain can be any naturally occurring variable domains or its engineered forms.Engineered forms meaning Refer to that Variable domain has been generated using recombinant DNA engineering technology.This kind of engineered forms include for example by specific It is inserted into the amino acid sequence of antibody, lacks or change or it is inserted into, lack or is changed, generated from specific antibodies variable region Those.Specific example includes such engineering Variable domain, contains at least one CDR and optionally one or more The remainder of amino framework acids from first antibody and the Variable domain from secondary antibody.

Variable domain can be covalently attached at least one other antibody domain or its segment in C-terminal amino acid. Thus, for example, being present in the V in Variable domain_HStructural domain can connect with immunoglobulin CH1 structural domain or its segment It connects.Similarly, V_LStructural domain can be with C_KStructural domain or the connection of its segment.Such as in this way, antibody can be Fab segment, Wherein antigen-binding domains contain its C-terminal respectively with CH1 structural domain and C_KThe V for the association that structural domain is covalently attached_HStructure Domain and V_LStructural domain.CH1 structural domain can be extended with further amino acid, such as, it is desirable to provide as present in Fab segment A part of hinge area or hinge region structural domain is intended to provide other structures domain, such as antibody CH2 structural domain and CH3 structural domain.

7. composition

The present invention also provides composition, including pharmaceutical composition, the composition includes as the above and elsewhere this paper is extensive The chimeric polyeptides or complex of description or the nucleic acid construct that can therefrom express chimeric polyeptides or complex.Representative compositions can With the buffer selected comprising the required purposes according to chimeric polyeptides or complex, and can also be comprising being suitable for desired use Other substances.In the case where desired use is induction immune response, composition is referred to as " immunogenicity " or " immunological regulation Property " composition.This kind of composition includes prophylactic compositions (that is, the group applied for the purpose of prevention disease (such as infection) Close object) and therapeutic composition (that is, the composition applied for the purpose for the treatment of disease (such as infection)).It therefore can be for Prevent, mitigate, treating the symptoms or therapeutic purposes, immune regulative composition of the invention is applied to recipient.

Those skilled in the art can select the Suitable buffer suitable for desired use easily, wherein known in the art more The buffer of seed type.In some cases, composition may include pharmaceutically acceptable excipient, many of described pharmaceutically acceptable Excipient is known in the art and without being discussed at length here.Pharmaceutically acceptable tax has been fully described in a variety of publications Shape agent, the publication is for example including A.Gennaro (2000) " Remington:The Science and Practice of Pharmacy ", the 20th edition, Lippincott, Williams, &Wilkins；Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C.Ansel et al. writes, the 7th addendum, Lippincott, Williams, & Wilkins；It is write with Handbook of Pharmaceutical Excipients (2000) A.H.Kibbe et al., the 3rd increases Mending Amer.Pharmaceutical Assoc.

In some embodiments, composition includes more than one (that is, different) chimeric polyeptides of the present invention or complex (for example, chimeric polyeptides, respective extracellular domain polypeptide corresponds to different enveloped virus fusion proteins) is a kind of or more Kind can therefrom express the nucleic acid construct of chimeric polyeptides or complex.

Pharmaceutical composition of the invention may be at fitting through the form of injection application, in the system for being suitable for being orally ingested Agent (for example, capsule, tablet, pastille, elixir) is in ointment, cream or the lotion form for being suitable for local application It is suitable as the form of eye drops delivering, is sprayed in sucking (as by nasal inhalation or oral sucking) application is fitted through Dosage form formula or the form applied in suitable parenteral (that is, subcutaneous, intramuscular or intravenous injection).

In the pharmaceutical composition that complementarity effective component such as adjuvant or biologically instrumentality can also be incorporated herein. Although adjuvant can be included in pharmaceutical composition of the invention, these pharmaceutical compositions are not needed inevitably comprising adjuvant.At this It, can be to avoid the reactionogenicity problem being originated from using adjuvant in the case of kind.

In general, the adjuvanticity under pharmaceutical composition background of the invention includes but is not limited to (quantitative or qualitative) enhancing The ability for the immune response that immunogenic components (for example, chimeric polyeptides or complex of the invention) induce in composition.This can To be reduced to the dosage of immunogenic components required by generating immune response or level and/or be reduced to immune needed for generating answer Answer required immunity inoculation times or frequency.

Any suitable adjuvant can be included in pharmaceutical composition of the invention.For example, can use the adjuvant based on aluminium. Appropriate adjuvants based on aluminium include but is not limited to aluminium hydroxide, aluminum phosphate and combinations thereof.European patent number 1216053 and the U.S. are special Other specific examples of the utilizable adjuvant based on aluminium are described in benefit number 6,372,223.Other suitable adjuvants include Incomplete Freund's adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.)；Merck adjuvant 65 (Merck and Company,Inc.,Rahway,N.J.)；AS-2(SmithKline Beecham,Philadelphia,Pa.)；Aluminium salt Such as gel aluminum hydroxide (alum) or aluminum phosphate；The salt of calcium, iron or zinc；The insoluble suspension of acylated tyrosine；Acylated sugar；Sun from The polysaccharide of submode or anion mode derivatization；Polyphosphazene；Biodegradable microspheres；Monophosphoryl lipid A and quil A；Water Packet fat liquor is included in european patent number 0399843, U.S. Patent number 7,029,678 and PCT Publication WO 2007/006939 Described in those；And/or additional cell factor, such as GM-CSF or proleulzin, -7 or -12, granulocytes-macrophages collection G-CSF (GM-CSF), tumor necrosis factor (TNF), monophosphoryl lipid A (MPL), cholera toxin (CT) or its composition are sub- Base, heat labile enterotoxin (LT) or its composition subunit, toll sample receptors ligand adjuvant, such as lipopolysaccharides (LPS) and its derivative (for example, monophosphoryl lipid A and 3- deacetylation monophosphoryl lipid A), flavivirus NS 1 and muramyl dipeptide (MDP).

Pharmaceutical composition of the invention can be provided in kit.Kit may include additional component to assist reality Apply method of the invention, the additional component such as application device, buffer and/or diluent.Kit may include holding Receive various components container and in the methods of the invention use reagent constituents specification.

8. dosage and administration method

Composition is with " effective quantity " (that is, the amount for realizing expected purpose effectively in subject) application.It is applied to patient's The dosage of reactive compound should be enough to realize in subject over time beneficial to reaction, such as at least one related to infection Symptom reduce.The amount or administration frequency of pharmaceutical active compounds to be administered can depend on subject to be treated, including Age, gender, weight and its general health.In this respect, will depend on for the precise volume of the reactive compound of application In the judgement of practitioner.By routine experiment, those skilled in the art will determine chimeric polyeptides as described herein or compound Effective, the nontoxic amount of body, to be included in the pharmaceutical composition of the present invention for required treatment results.

In general, pharmaceutical composition of the invention can according to administration method and recipient's physical trait (including healthy shape State) compatible mode is applied and is applied in such a manner, so that it excites required effect (that is, treating effective, immunogenicity And/or protectiveness effect).For example, the optimal dose of pharmaceutical composition of the present invention can depend on many factors, including but unlimited Single medicine or complementary therapy whether are being used as in the physical trait (for example, age, weight, gender) of subject, compound Use, the progress (that is, pathological state) of the MHC Limit Type of patient, virus infection and can be by those skilled in the art The other factors of identification.Such as in Gennaro (2000) " Remington:The Science and Practice of Pharmacy ", the 20th edition, Lippincott, Williams, &Wilkins；With Gilman et al. (writing), (1990), “Goodman And Gilman's:The Pharmacological Bases of Therapeutics”,Pergamon Admissible a variety of general attention items when the optimal dose for determining pharmaceutical composition of the present invention are described in Press.

In some embodiments, theme chimeric polyeptides or complex or the core of chimeric polyeptides or complex can therefrom be expressed The effective quantity of acid con-struct is such amount, is enough preventative or therapeutic effect needed for realizing, for example, reducing related to infection Symptom and/or reduce individual in infective agent number.In these embodiments, it is treated with unused chimeric polyeptides or complex Individual in symptom or infective agent number when comparing, effective quantity reduce symptom relevant to infection and/or reduction it is individual in passes Contaminate the number of object up at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, or more.The symptom and measurement infected caused by pathogenic organisms The method of this kind of symptom is known in the art.The method of pathogenic organisms number is standard in this field in measurement individual.

In some embodiments, theme chimeric polyeptides or complex or the core of chimeric polyeptides or complex can therefrom be expressed The effective quantity of acid con-struct is such amount, and effectively excitation is for enveloped virus fusion protein under the administration method of selection Immune response.

In some embodiments, for example, " effective quantity " is effective in the case that wherein chimeric polyeptides include heterologous antigen Promote excitation for the amount of the immune response of antigen.For example, being from the cause with derivative extracellular domain polypeptide in heterologous antigen When the antigen of the different pathogenic organisms of diease occurrence object, theme chimeric polyeptides or complex or chimeric polyeptides or complex can be therefrom expressed The effective quantity of nucleic acid construct be such amount, effectively excitation for the antigen immune response and preferably effectively protect Protect infection or symptom relevant to infection caused by host versus pathogenic organisms.In these embodiments, with it is unused chimeric more When comparing, effective quantity is reduced and infection phase caused by pathogenic organisms for peptide or symptom in the individual of complex treatment or infective agent number The symptom of pass and/or reduce correspond in individual the number of the infective agent of pathogenic organisms up at least about 10%, at least about 20%, extremely Few about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, Or more.The method of the symptom and this kind of symptom of measurement that infect caused by pathogenic organisms is known in the art.

Alternatively, in the case where heterologous antigen is related cancer or tumor associated antigen, theme chimeric polyeptides or complex Or can therefrom express the effective quantity of the nucleic acid construct of chimeric polyeptides or complex is such amount, in the administration method of selection Effective immune response down, the immune response effectively reduce or inhibit cancer or growth of tumour cell, reduce cancer or tumour cell A possibility that agglomerate or cancer or tumor cell number or reduction cancer or tumour will be formed.In these embodiments, with it is unused When comparing, effective quantity reduces tumour for chimeric polyeptides or tumour growth in the individual of complex treatment and/or tumor cell number Growth and/or tumor cell number up at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, or more.Measure tumour growth and tumour The method of cell number is known in the art.

The amount of chimeric polyeptides or complex in each dosage is selected as such amount, born of the same parents of the amount induction for coding The immune response of extracellular portion polypeptide and/or the reaction of induction immunoprotection or other immunization therapies reaction, at the same not usually with The relevant obvious adverse side effect of common vaccine.This amount will depend on which kind of specific extracellular domain polypeptide, vaccine system used Whether agent includes adjuvant and a variety of Host Dependence factors and changes.

Pharmaceutical composition of the invention can pass through standard way, including but not limited to parenteral (for example, intravenous) way Diameter is applied to recipient.

Pharmaceutical composition of the invention can be additional alone or in combination medicine be applied to recipient together.In medicine group In the embodiment for closing synchronous with the medicine application of object, apply can be simultaneously or sequentially (that is, apply pharmaceutical composition, with After apply the drug, or vice versa).

Generally, in therapeutic application, treatment can be carried out in the duration of morbid state or situation.In addition, ability Domain those of ordinary skill is evident that the optimised quantity of each dosage and interval will depend on the disease for receiving to treat The property and degree of state or symptom, administration form, approach and position and the speciality for receiving the specific individual treated.It can be with Optimum condition is determined using routine techniques.

(for example, prophylactic use) in many cases, it may be desirable to several times or multiple applications pharmaceutical composition of the invention Object.For example, pharmaceutical composition 1,2,3,4,5,6,7,8,9,10, secondary or more time can be applied.Application can be spaced about one week To about 12 weeks, and in certain embodiments, application can be spaced about one Zhou Zhiyue surrounding.It is being repeated exposure to the present invention In the case where the special pathogen or other diseases related component of pharmaceutical composition targeting, it may be necessary to periodically apply the present invention again Pharmaceutical composition.

Those of ordinary skill in the art will also be apparent that, the conventional course for the treatment of can be used and determine that test, determination are most preferably applied Use process.

In the case where two or more entities " combination " or " synchronization " are applied to subject, they can be at single group It closes and is administered simultaneously in object or is administered simultaneously in discrete composition or applied in discrete composition in the separated time.

Certain embodiments of the present invention are related to by multiple discrete dosage application pharmaceutical compositions.Therefore, prevention (connects Kind) and the method for the treatment of infection described herein cover to the multiple discrete dosage of subject's application, for example, model in a limited time period Enclose interior application.Therefore, the method for prevention (being inoculated with) and treatment infection disclosed herein includes application priming dose (priming Dose pharmaceutical composition of the present invention).Priming dose can be followed by booster.Reinforcing agent can be used for inoculating purpose.In In multiple embodiments, at least once, twice, three times or more by pharmaceutical composition or vaccine administration.

The method for measuring immune response is known to persons of ordinary skill in the art.Illustrative methods include that solid-phase heterogeneous is surveyed Determine method (for example, enzyme-linked immunosorbent assay), molten liquid phase assays (for example, electrochemical luminescence measuring method), amplification shine it is close Journey homogeneous assay method, flow cytometry, intracellular cytokine dyeing method, function T cell measuring method, function B cell measuring method, function It can Monocyte-macrophages measuring methods, tree-shaped and reticuloendothelial cell measuring method, measurement NK cell effect, immunocyte IFN-γ generates, viral RNA/DNA is quantitative (for example, in serum or other body fluid or tissue/organ in tissue or biological fluid Viral RNA or DNA's quantifies), oxidative burst measuring method, cell toxicant specific cytolytic measuring method, pentamer binding assay Method and phagocytosis are assessed with apoptosis.

Skilled artisans will appreciate that can to invention as shown in the specific embodiments carry out it is numerous variation and/or Modification is without departing from the spirit and scope of the present invention such as fully described.Therefore, embodiment of the present invention should be in terms of whole It is considered as illustrative and not restrictive.

In order to be readily appreciated that the present invention and generate actual effect, will be described now by following non-limiting embodiment Specific preferred embodiment.

Embodiment