Isolated epitope polypeptide
阅读说明：本技术 一种分离的抗原表位多肽 (Isolated epitope polypeptide ) 是由 肖鹤 冯健男 沈倍奋 陈楠 陈国江 王晶 乔春霞 李新颖 刘成华 于 2021-10-09 设计创作，主要内容包括：本发明公开了一种分离的抗原表位多肽,所述抗原表位多肽包含SEQ IDNO.1所示的氨基酸序列或其变体,SEQ ID NO.3所示的氨基酸序列或变体,或SEQ ID NO.2所示的氨基酸序列。本发明的抗原表位多肽可特异性结合西尼罗河病毒抗体,利用该性质,可开发检测西尼罗河病毒抗体的试剂盒以及预防西尼罗河病毒感染的疫苗。(The invention discloses an isolated epitope polypeptide, which comprises an amino acid sequence shown in SEQ ID NO.1 or a variant thereof, an amino acid sequence shown in SEQ ID NO.3 or a variant thereof, or an amino acid sequence shown in SEQ ID NO. 2. The epitope polypeptide can be specifically combined with a west nile virus antibody, and by utilizing the property, a kit for detecting the west nile virus antibody and a vaccine for preventing west nile virus infection can be developed.)
1. An isolated epitope polypeptide comprising a polypeptide fragment of west nile virus E protein or a variant thereof.
2. The epitope polypeptide of claim 1, wherein said epitope polypeptide comprises the amino acid sequence shown in SEQ ID No.1 or a variant thereof, the amino acid sequence shown in SEQ ID No.3 or a variant thereof, or the amino acid sequence shown in SEQ ID No. 2;
preferably, the variant of SEQ ID No.1 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.1 is replaced;
2) the 3 rd position of the amino acid sequence shown in SEQ ID NO.1 is replaced;
3) the 5 th position of the amino acid sequence shown in SEQ ID NO.1 is replaced;
preferably, the variant of SEQ ID No.3 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
2) the 2 nd position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
3) the 6 th position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
preferably, the variant of SEQ ID No.1 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A;
2) the 3 rd position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A;
3) the 5 th position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A;
preferably, the variant of SEQ ID No.3 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.3 is replaced by A;
2) the 2 nd position of the amino acid sequence shown in SEQ ID NO.3 is replaced by A;
3) the amino acid sequence shown in SEQ ID NO.3 has a substitution of A at position 6.
3. An antigenic polypeptide comprising a variant of the antigenic epitope polypeptide of claim 1; preferably, the sequence of the antigen polypeptide is shown in SEQ ID NO.7-SEQ ID NO. 18.
4. A complex comprising the epitope polypeptide of claim 1 or 2;
preferably, the complex further comprises a carrier which forms a complex with the epitope polypeptide by means of coupling, conjugation or fusion;
preferably, the coupling includes MBS method, glutaraldehyde method, active ester method, carbodiimide method, halogenated nitrobenzene method, imidic acid ester method;
preferably, the carrier comprises a protein, toxin or lipid;
preferably, the proteins include human serum albumin, bovine thyroglobulin, keyhole limpet blood albumin and other gamma globulins;
preferably, the complex is a fusion protein;
preferably, the fusion protein is formed by connecting the antigenic epitope polypeptide of claim 1 or 2 or the antigenic polypeptide of claim 3 with the N-terminal of Fc;
preferably, the amino acid sequence of Fc is shown in SEQ ID NO. 5.
5. A viroid particle displaying the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3 or the complex of claim 4.
6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, or the complex of claim 4, or a nucleic acid vector comprising the same.
7. A host cell comprising the nucleic acid molecule of claim 6 or a nucleic acid vector comprising the same.
8. A product comprising any one of:
1) an antibody that specifically binds to the epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, the complex of claim 4; preferably, the antibody is a monoclonal or polyclonal antibody;
2) a composition comprising any one of:
a) a pharmaceutical composition comprising the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, the complex of claim 4, the viroid-like particle of claim 5, the nucleic acid molecule of claim 6 or a nucleic acid vector comprising the same, the antibody; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is a vaccine;
b) a detection composition comprising the epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, the complex of claim 4, the antibody; preferably, the detection composition further comprises a detectable moiety.
9. A method for detecting antibodies against west nile virus E protein for non-diagnostic purposes, the method comprising using the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, or the complex of claim 4; preferably, the method comprises the steps of:
1) obtaining a sample containing an antibody against West Nile virus E protein;
2) contacting the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3 or the complex of claim 4 with the sample of step 1);
3) detecting an antigen-antibody reaction;
preferably, the antibody comprises the following sequence: the CDR sequences of the heavy chain variable region are respectively shown in SEQ ID NO. 19-21; CDR sequences of the light chain variable region are respectively shown in SEQ ID NO. 22-24;
preferably, the antibody comprises the following sequence: the heavy chain variable region sequence is shown in SEQ ID NO. 25; the light chain variable region sequence is shown in SEQ ID NO. 26.
10. An application, the application comprising any one of:
1) use of the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3 or the complex of claim 4 as an immunizing antigen;
2) use of the antigenic epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3 or the complex of claim 4 for the preparation of an antibody;
3) use of the epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, or the complex of claim 4 for the preparation of a vaccine for the prevention of west nile virus infection;
4) use of the epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, or the complex of claim 4 for the preparation of an antibody for detecting an E protein of west nile virus;
5) use of the epitope polypeptide of claim 1 or 2, the antigenic polypeptide of claim 3, or the complex of claim 4 for the preparation of a medicament for the treatment of west nile virus infection;
6) use of the viroid particle of claim 5, the nucleic acid molecule of claim 6 or a nucleic acid vector comprising the same for the preparation of a vaccine for the prevention of west nile virus infection;
7) use of the viroid particle of claim 5, the nucleic acid molecule of claim 6 or a nucleic acid vector comprising the same for the preparation of a medicament for the treatment of west nile virus infection.
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a separated epitope polypeptide.
Background
West Nile Virus (WNV) infection can cause West Nile fever, West Nile viral encephalitis and meningitis, and is a zoonosis, natural epidemic source and acute infectious disease. Since the outbreak of WNV in new york, usa in 1999, the virus has spread rapidly to many countries and regions of the world, becoming a viral disease that seriously threatens human health, and has attracted public health world concerns. WNV can infect many kinds of mosquitoes and birds, and spread viruses along the migration path of the birds through the alternate infection of the mosquitoes and the birds; mosquitoes can also infect WNV to a variety of mammals, such as humans, horses, dogs, cats, and poultry, such as chickens, geese, etc., by biting WNV-infected birds; the virus can also be transmitted vertically by blood transfusion, organ transplantation, lactation and placenta. At present, two million people are infected with WNV globally, the morbidity is over 20 percent, and compared with the prior WNV infection, the severe cases are obviously increased, and the mortality rate is increased to 5 to 15 percent.
Epitopes are chemical groups, also called antigenic determinants, present on the surface of an antigen that determine the specific structure of the antigen. The antigen is combined with the receptor on the surface of the corresponding lymphocyte through the epitope, so that the lymphocyte is activated to cause the immune response. A single antigenic molecule can have one or more different epitopes, each epitope being of only one antigenic specificity. Therefore, an epitope is a target structure recognized by immune cells, is also a basis for specificity of immune reaction, and generally comprises 5 to 7 amino acids and monosaccharide residues, and at most, does not exceed 20 amino acid residues.
The development of the epitope polypeptide is beneficial to preparing products for detecting West Nile virus antibodies, for example, the identified epitope synthetic polypeptide can be singly or mixedly coated on an ELISA plate, and can be used for detecting the virus antibodies. The antigen epitopes can be used for designing genetic engineering vaccines or for detecting West Nile virus and West Nile virus antibodies.
Disclosure of Invention
According to one aspect of the present invention, there is provided an isolated epitope polypeptide comprising a polypeptide fragment of west nile virus E protein or a variant thereof.
Further, the epitope polypeptide comprises an amino acid sequence shown in SEQ ID NO.1 or a variant thereof, an amino acid sequence shown in SEQ ID NO.3 or a variant thereof, or an amino acid sequence shown in SEQ ID NO. 2.
Preferably, the variant of SEQ ID No.1 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.1 is replaced;
2) the 3 rd position of the amino acid sequence shown in SEQ ID NO.1 is replaced;
3) the 5 th position of the amino acid sequence shown in SEQ ID NO.1 is replaced.
Preferably, the variant of SEQ ID No.1 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A;
2) the 3 rd position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A;
3) the 5 th position of the amino acid sequence shown in SEQ ID NO.1 is replaced by A.
Preferably, the variant of SEQ ID No.3 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
2) the 2 nd position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
3) the 6 th position of the amino acid sequence shown in SEQ ID NO.3 is replaced;
preferably, the variant of SEQ ID No.3 comprises any of:
1) the 1 st position of the amino acid sequence shown in SEQ ID NO.3 is replaced by A;
2) the 2 nd position of the amino acid sequence shown in SEQ ID NO.3 is replaced by A;
3) the amino acid sequence shown in SEQ ID NO.3 has a substitution of A at position 6.
According to another aspect of the present invention there is provided an antigenic polypeptide comprising a variant of an antigenic epitope polypeptide as hereinbefore described.
According to another aspect of the present invention there is provided a complex comprising an antigenic epitope polypeptide as hereinbefore described or an antigenic polypeptide as hereinbefore described.
Further, the complex also comprises a carrier, and the carrier forms a complex with the epitope polypeptide by means of coupling, conjugation or fusion.
Preferably, the coupling includes MBS method, glutaraldehyde method, active ester method, carbodiimide method, halogenated nitrobenzene method, imidic acid ester method.
Preferably, the carrier comprises a protein, toxin or lipid.
Preferably, the proteins include human serum albumin, bovine thyroglobulin, keyhole limpet blood albumin and other gamma globulins.
Preferably, the complex is a fusion protein.
In a specific embodiment of the present invention, the complex is the aforementioned antigenic epitope polypeptide or a fusion protein formed by connecting the aforementioned antigenic polypeptide to the N-terminus of Fc.
Specifically, the epitope polypeptide or the antigenic polypeptide is linked to the N-terminus of Fc via GGGGS.
The amino acid sequence of the Fc used in the invention is shown in SEQ ID NO. 5.
According to a further aspect of the invention there is provided a viroid displaying an antigenic epitope polypeptide, antigenic polypeptide or a complex as hereinbefore described.
According to a further aspect of the invention there is provided an isolated nucleic acid molecule or a nucleic acid vector comprising the same, comprising a nucleic acid sequence encoding an antigenic epitope polypeptide, antigenic polypeptide or complex as hereinbefore described.
According to a further aspect of the invention, there is provided a host cell comprising a nucleic acid molecule as hereinbefore described or a nucleic acid vector comprising the same.
The term "vector" as used herein refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted to allow expression of the protein. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma vacuolium viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.
Host cells useful in the present invention are prokaryotic cells. More preferably, the host cell is e. In a related embodiment, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of a protist cell, an animal cell (such as mammalian cell, avian cell and insect cell), a plant cell and a fungal cell. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS; or a fungal cell, such as a yeast cell, e.g., Saccharomyces cerevisiae; or insect cells such as Sf 9. According to a further aspect of the invention, there is provided an antibody which specifically binds to an epitope polypeptide as defined above, or a complex as defined above.
The vector may be transformed, transduced or transfected into a host cell by methods conventional in the art, such as chemical transformation by calcium chloride, high-voltage shock transformation, preferably shock transformation.
The epitope peptide of the present invention can be isolated and purified from a host cell using a method commonly used in the art. For example, the epitope peptide can be purified by centrifugation of the culture medium and the recombinant host cells, high-pressure homogenization for cell disruption, centrifugation for cell debris removal, and affinity chromatography. For the isolation and purification of the resulting product, purity identification can be performed using a method commonly used in the art. For example, Coomassie blue method, Kjeldahl method, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, antigen-antibody method, electrophoresis (for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis), sedimentation analysis, diffusion analysis, isotachy method, protein mass spectrometry, and the like.
The invention also provides a product, which comprises the antibody and the composition.
According to a further aspect of the invention, there is provided an antibody which specifically binds to an epitope polypeptide as defined above, an antigenic polypeptide as defined above, a complex as defined above; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
According to yet another aspect of the present invention, there is provided a composition comprising any one of:
1) a pharmaceutical composition comprising an antigenic epitope polypeptide as defined above, an antigenic polypeptide as defined above, a complex as defined above, a viroid as defined above, a nucleic acid molecule as defined above or a nucleic acid vector comprising same, an antibody as defined above.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is a vaccine.
2) A detection composition comprising the epitope polypeptide, the antigenic polypeptide, the complex, and the antibody; preferably, the detection composition further comprises a detectable moiety.
Detectable moieties include, for example, labels or tags. In some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. The imaging agent comprises a radioisotope and the contrast agent comprises iodine, gadolinium or iron oxide. The enzyme comprises horseradish peroxidase, alkaline phosphatase or beta-galactosidase. In some embodiments, the fluorescent label comprises Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2(RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridinium (methylacridinum) derivative. In some embodiments, the tag comprises AlexaMarkers, such as Alex680 or Alexa750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.
The term "label" as used herein refers to the incorporation of a detectable label, e.g., by the incorporation of a radiolabeled amino acid or with avidin (e.g., containing fluorescence detectable by optical or calorimetric methods) that can be labeledLabeled or enzymatically active streptavidin) to the biotin moiety. In some cases, the marker or markers may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,3H、14C、15N、35S、90Y、99Tc、111In、125I、131I) fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites for a second antibody, metal binding domains, epitope tags). In some embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance.
According to a further aspect of the invention there is provided a method for detecting antibodies against the E protein of West Nile Virus, which method comprises the use of an epitope polypeptide as hereinbefore described, an antigenic polypeptide as hereinbefore described or a complex as hereinbefore described, for non-diagnostic purposes.
Preferably, the method comprises the steps of:
1) obtaining a sample containing an antibody against West Nile virus E protein;
2) contacting the epitope polypeptide, the antigenic polypeptide or the complex with the sample of step 1);
3) detecting an antigen-antibody reaction;
preferably, the antibody comprises the following sequence: the CDR sequences of the heavy chain variable region are respectively shown in SEQ ID NO. 19-21; the CDR sequences of the light chain variable region are respectively shown in SEQ ID NO. 22-24.
Preferably, the antibody comprises the following sequence: the heavy chain variable region sequence is shown in SEQ ID NO. 25; the light chain variable region sequence is shown in SEQ ID NO. 26.
According to a further aspect of the invention, there is provided a use comprising any one of:
1) use of an epitope polypeptide as defined above, an antigenic polypeptide as defined above or a complex as defined above as an immunizing antigen;
2) use of an epitope polypeptide as defined above, an epitope polypeptide as defined above or a complex as defined above for the preparation of an antibody;
3) the application of the antigen epitope polypeptide, the antigen polypeptide or the compound in preparing a vaccine for preventing West Nile virus infection;
4) the application of the antigen epitope polypeptide, the antigen polypeptide or the compound in preparing an antibody for detecting the E protein of the West Nile virus;
5) the use of the epitope polypeptide, the antigenic polypeptide or the complex for the manufacture of a medicament for the treatment of West Nile Virus infection;
6) use of a viroid as hereinbefore described, a nucleic acid molecule as hereinbefore described or a nucleic acid vector comprising the same for the preparation of a vaccine for the prevention of west nile virus infection;
7) use of a viroid as hereinbefore described, a nucleic acid molecule as hereinbefore described or a nucleic acid vector comprising the same for the manufacture of a medicament for the treatment of west nile virus infection.
In the present application, the term "epitope polypeptide" refers to any polypeptide fragment that can be recognized by a specific antibody. In particular embodiments, the "epitope polypeptide" may be 4-25 amino acids, such as 9-25 amino acids, 9-20 amino acids, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids in length.
In this application, "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen from which the antibody is derived. Here, the binding affinity of an antibody that binds a first antigen to a second antigen is undetectable or even weak if detectable.
In the present invention, methods that can be used for detection of West Nile Virus infection include, but are not limited to, for example, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition, immunohistochemical assay, and the like.
Suitable pharmaceutically acceptable carriers include, for example, one or more of water, physiological saline, phosphate buffer, levulose, glycerol, ethanol, and the like, as well as combinations thereof. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide, antibody or nucleic acid molecule.
Modes of administration of the complexes or pharmaceutical compositions of the invention include, but are not limited to, conventional routes of administration, such as intravenous drip, intramuscular injection, vaginal, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powders, ointments or drops), or nasal routes, and the like. In particular, the administration form is an injection or infusion form.
The complexes or pharmaceutical compositions of the invention may be in various forms including, but not limited to, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, solutions, dispersions or suspensions, liposomes, suppositories, solutions for injection and infusion. The preferred form depends on the particular mode of administration and its prophylactic, therapeutic or diagnostic use.
Pharmaceutical compositions of the invention suitable for parenteral injection may contain sterile aqueous or nonaqueous solutions, aerosols, suspensions or emulsions as required for pharmaceutical preparations, or sterile powders which may be resuspended in injectable solutions or aerosols at the time of use. Such as suitable aqueous and non-aqueous vehicles, vehicles and diluents such as water, ethanol, polyols (e.g., propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures, rape oil (e.g., olive oil), and organic fats which may be used for injection, such as oleic acid, e.g., with lecithin shells to maintain proper fluidity of the drug, e.g., with aerosols, surfactants to maintain proper particle size.
The pharmaceutical compositions of the present invention may also contain adjuvants which provide protection, moisturization, emulsification and aerosolization, as well as fast dissolving ingredients to prevent microbial contamination, such as various antibacterial agents, antifungal agents, such as methylparaben, chlorobutanol, phenol, sorbic acid and the like. Agents for maintaining osmotic pressure, such as sugars, NaCl, and the like, may also be included. Prolonged adsorption of pharmaceutical ingredients for injection may be achieved by using agents that prolong adsorption, such as monostearate salts and gels.
The vaccine comprising the epitope peptide of the present invention can be prepared by a method known in the art. For example, as the vaccine, there are an injection, a solid preparation, and the like containing the epitope peptide of the present invention as an active ingredient. The epitope peptide may be formulated in a neutral or salt form, and examples of the pharmaceutically acceptable salt include inorganic salts such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid and tartaric acid. Further, if necessary, an auxiliary agent such as albumin, a wetting agent, an emulsifier, or the like may be added.
The vaccines of the present invention are administered in a therapeutically effective amount. The dose to be administered depends on the subject to be treated and the immune system, and the necessary dose is determined at the discretion of the clinician. The administration interval may be set according to the object or purpose.
The epitope polypeptide, the antibody or the pharmaceutical composition can be combined with other antiviral agents to be used for preventing and/or treating West Nile virus infection and diseases related to the West Nile virus infection. The epitope polypeptide, antibody or pharmaceutical composition of the present invention can be administered simultaneously, separately or sequentially with other antiviral agents. Other antiviral agents include, but are not limited to, for example, ribavirin, adamantane, carboxyurea, IL-2, IL-12, and pentacarboxocytic acid.
Drawings
FIG. 1 shows an SDS-PAGE electrophoresis of epitope peptide-Fc fusion proteins;
FIG. 2 is a graph showing the results of detection of the binding specificity of the epitope peptide-Fc fusion protein to an antibody, wherein A: epitope peptide 1-Fc; b: epitope peptide 2-Fc; c: epitope peptide 3-Fc;
FIG. 3 shows a SDS-PAGE electrophoresis of WNV EDIII mutant-Fc fusion proteins, wherein A: carrying out multipoint mutation; b: single point mutation 1/2/3/4/6/8/9; c: single point mutation 5/7/10;
figure 4 shows a graph of the detection results of the binding specificity of WNV EDIII mutant-Fc fusion protein to an antibody, wherein a: carrying out multipoint mutation; b: single point mutation;
fig. 5 shows a graph of the results of epitope competition experiments, where a: carrying out multipoint mutation; b: single point mutation;
Detailed Description
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1 epitope peptide fusion protein preparation and functional assays
Preparation of antigen epitope peptide fusion protein
1. Method of producing a composite material
1.1 preparation of fusion protein expression vectors
Epitope peptide 1: QYTGTD (SEQ ID NO.1)
Epitope peptide 1-Fc
Epitope peptide 2: HGTVVLELQYTGTDGPCKVP (SEQ ID NO.2)
Epitope peptide 2-Fc
Epitope peptide 3: TYGVCSKAF (SEQ ID NO.3)
Epitope peptide 3-Fc
WNV EDIII：(SEQ ID NO.4)
WNV EDIII-Fc
The method comprises the following steps: the epitope peptide and Fc fusion sequence are amplified by PCR method overlap, GGGGS is introduced between epitope peptide or WNV EDIII and Fc sequence (the amino acid sequence of Fc is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO. 6), and the coding sequence of the fusion fragment is cloned into an expression vector by molecular cloning method.
1.2 transfecting the expression vector into a mammalian cell for expression.
1.3 the expression supernatant was collected and purified by using Protein A FF Protein column from GE.
1.4 elution with citrate buffer pH3.0, collection of the effluent, and immediately using 1mol/L pH 8.5TRIS-HCL buffer neutralization, pH7.2, 0.01mol/L PBS dialysis for 72h, 0.22 u m filter sterilization.
2. Results
The expression and purification results of the fusion protein are shown in figure 1, and the epitope peptide 1-Fc, the epitope peptide 2-Fc and the epitope peptide 3-Fc are successfully expressed.
Second, detection of antigen-antibody binding specificity
2.1 method
WNV-XH1 antibody was detected by ELISA assay (sequence of WNV-XH1 in 202011075191X). Binding to an epitope peptide. The specific experimental steps are as follows:
1) coating 10 mu g/ml of epitope peptide 1-Fc, epitope peptide 2-Fc and epitope peptide 3-Fc fusion protein in an ELISA plate, and standing overnight at 4 ℃;
2) blocking unbound sites with skim milk powder, then washing 3 times with PBS buffer containing 0.1% tween;
3) preparing antibodies WNV-XH1 with different concentrations, adding the antibodies to the ELISA plate bar in the step 2, incubating for 1h at 37 ℃, and washing for 3 times by using PBS buffer solution containing 0.1% Tween;
4) addition of HRP-labeled goat anti-human IgG (Fab')2Antibody, incubated at 37 ℃ for 30min, and incubated withWashing with PBS buffer solution containing 0.1% Tween for 3 times; color development was performed by adding TMB, and OD was measured after 1mol/L sulfuric acid was terminated450The value of (c).
2.2 results
The results are shown in FIG. 2, which indicates that epitope peptide 1-Fc, epitope peptide 2-Fc, and epitope peptide 3-Fc can specifically bind to WNV-XH1 antibody.
Example 2 Effect of epitope peptide mutations on binding Capacity
Firstly, preparation of WNV EDIII mutant fusion protein
1. Method of producing a composite material
1.1 mutation design
Single-point and multi-point mutation is carried out on the epitope peptide 1: QYTGTD on WNV EDIII, and the mutated polypeptides are shown as follows:
WNV EDIII MUT2：(SEQ ID NO.7)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELAAAGAAGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANAKVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT6：(SEQ ID NO.8)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELAY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT7：(SEQ ID NO.9)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELQA TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT8：(SEQ ID NO.10)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELQY AGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT9：(SEQ ID NO.11)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELQY TGADGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT10：(SEQ ID NO.12)
EKLQLKG TTYGVCSKAF KFLGTPADTG HGTVVLELQY TGTAGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
the epitope peptide 3: TYGVCSKAF is subjected to single-point and multi-point mutation, and the mutated polypeptide is shown as follows:
WNV EDIII MUT1：AAGVCAAAA(SEQ ID NO.13)
EKLQLKG TAAGVCAAAA KFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT1：AYGVCSKAF(SEQ ID NO.14)
EKLQLKG TAYGVCSKAF KFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT2：TAGVCSKAF(SEQ ID NO.15)
EKLQLKG TTAGVCSKAF KFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT3：TYGVCAKAF(SEQ ID NO.16)
EKLQLKG TTYGVCAKAF KFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT4：TYGVCSAAF(SEQ ID NO.17)
EKLQLKG TTYGVCSAAF KFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANA KVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
WNV EDIII SMUT5：TYGVCSKAA(SEQ ID NO.18)
EKLQLKG TTYGVCSKAAKFLGTPADTG HGTVVLELQY TGTDGPCKVP ISSVASLNDL TPVGRLVTVN PFVSVATANAKVLIELEPPF GDSYIVVGRG EQQINHHWHK SGSSIG
1.2 methods
1) And (3) completely synthesizing the WNV EDIII mutant sequence gene.
2) Amplifying WNV EDIII mutants and Fc fusion sequences by using a PCR method overlap, introducing GGGGS between the WNV EDIII mutants and the Fc sequences, and cloning the fusion fragments into an expression vector by using a molecular cloning method.
3) The expression vector is transfected into mammalian cells for expression.
4) The expression supernatant was collected and purified by a Protein A FF Protein column from GE.
5) Eluting with citric acid buffer solution of pH3.0, collecting eluate, immediately neutralizing with 1mol/L TRIS-HCL buffer solution of pH 8.5, dialyzing with PBS of pH7.2 and 0.01mol/L for 72h, and filtering with 0.22 μm filter membrane for sterilization.
1.3 results
Protein expression and purification were performed by SDS-PAGE, and the results are shown in FIG. 3, and WNV EDIII-Fc and the above WNV EDIII mutant-Fc were successfully expressed, note: in the figure, 1-10 respectively represent WNV EDIII SMUT1-Fc-WNV EDIII SMUT10-Fc, MUT1 represents WNV EDIII MUT1-Fc, MUT2 represents WNV EDIII MUT2-Fc, and EDIII represents WNV EDIII-Fc.
Second, detection of antigen-antibody binding specificity
2.1 method
The combination condition of the WNV-XH1 antibody and WNV EDIII-Fc and the WNV EDIII mutant-Fc is detected by ELISA experiment. WNV-XH1 antibody sequences see the patent application No. 202011075191X.
1) Coating each WNV EDIII mutant-Fc 2. mu.g/ml in ELISA plates overnight at 4 ℃;
2) blocking unbound sites with skim milk powder, then washing 3 times with PBS buffer containing 0.1% tween;
3) preparing antibodies WNV-XH1 with different concentrations, adding the antibodies to the ELISA plate bar in the step 2, incubating for 1h at 37 ℃, and washing for 3 times by using PBS buffer solution containing 0.1% Tween;
4) addition of HRP-labeled goat anti-human IgG (Fab')2Antibody, 37 degrees C were incubated for 30min, with 0.1% Tween PBS buffer washing 3 times; color development was performed by adding TMB, and OD was measured after 1mol/L sulfuric acid was terminated450The value of (c).
2.2 results
The results are shown in FIG. 4, indicating that WNV EDIII-Fc, WNV EDIII SMUT1-Fc, WNV EDIII SMUT2-Fc, WNV EDIII SMUT3-Fc, WNV EDIII SMUT6-Fc, WNV EDIII SMUT8-Fc, WNV EDIII SMUT9-Fc specifically bind to WNV-XH1 antibodies. Note that in the figure, MUT1 represents WNV EDIII MUT1-Fc, MUT2 represents WNV EDIII MUT2-Fc, EDIII represents WNV EDIII-Fc, and SMUT1-SMUT10 respectively represent WNV EDIII SMUT1-Fc-WNV EDIII SMUT 10-Fc.
Third, epitope competition experiment
1. Method of producing a composite material
1) Coating 1. mu.g/ml WNV EDIII in ELISA plates overnight at 4 ℃;
2) blocking unbound sites with skim milk powder, then washing 3 times with PBS buffer containing 0.1% tween;
3) the corresponding mutant protein diluted by a blocking solution in a gradient manner with the initial concentration of 50 mug/mL is diluted by 2 times, mixed with the antibody WNV-XH1 with the corresponding concentration and placed in an incubator at 37 ℃ for 30 min. The enzyme-linked plate is added according to the amount of 100 mu L/hole, and the incubator is placed for 1h at 37 ℃.
4) Addition of HRP-labeled goat anti-human IgG (Fab')2Antibody, 37 degrees C were incubated for 30min, with 0.1% Tween PBS buffer washing 3 times; color development was performed by adding TMB, and OD was measured after 1mol/L sulfuric acid was terminated450The value of (c).
2. Results
The results are shown in FIG. 5, indicating that WNV EDIII SMUT4-Fc, WNV EDIII SMUT5-Fc, WNV EDIII SMUT7-Fc, WNV EDIII SMUT10-Fc, WNV EDIII MUT1-Fc, WNV EDIII MUT2-Fc did not bind to WNV-XH1 antibody. Note: WNV EDIII represents WNV EDIII-Fc, WNV EDIII MUT1 represents WNV EDIII MUT1-Fc, WNV EDIII MUT2 represents WNV EDIII MUT2-Fc, SMUT4, SMUT5, SMUT7 and SMUT10 represent WNV EDIII SMUT4-Fc, WNV EDIII SMUT5-Fc, WNV EDIII SMUT7-Fc and WNV EDIII SMUT10-Fc respectively.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> an isolated epitope polypeptide
<141> 2021-10-09
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Tyr Thr Gly Thr Asp
1 5
<210> 2
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
His Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro
1 5 10 15
Cys Lys Val Pro
20
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Thr Tyr Gly Val Cys Ser Lys Ala Phe
1 5
<210> 4
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Lys Leu Gln Leu Lys Gly Thr Tyr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 5
<211> 230
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
100 105 110
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Pro Gly Lys
225 230
<210> 6
<211> 690
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 60
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 120
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 180
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 240
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 300
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 360
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 420
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 480
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 540
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 600
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 660
cagaagagcc tctccctgtc tccgggtaaa 690
<210> 7
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Ala Ala Ala Gly Ala Ala Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 8
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Ala Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 9
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Ala Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 10
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 11
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Ala Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 12
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Ala Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 13
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Glu Lys Leu Gln Leu Lys Gly Thr Ala Ala Gly Val Cys Ala Ala Ala
1 5 10 15
Ala Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 14
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Glu Lys Leu Gln Leu Lys Gly Thr Ala Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 15
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Lys Leu Gln Leu Lys Gly Thr Thr Ala Gly Val Cys Ser Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 16
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ala Lys Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 17
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Ala Ala
1 5 10 15
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 18
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser Lys Ala
1 5 10 15
Ala Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val
20 25 30
Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile
35 40 45
Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg Leu Val
50 55 60
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys Val Leu
65 70 75 80
Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg
85 90 95
Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser Ser Ile
100 105 110
Gly
<210> 19
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Asp Tyr Trp Ile Glu
1 5
<210> 20
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Asp Ile Leu Tyr Gly Asn Gly Arg Thr Arg Tyr Asn Glu Lys Leu Lys
1 5 10 15
Gly
<210> 21
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Ser Ala Ser Tyr Gly Asp Tyr Ala Asp Tyr
1 5 10
<210> 22
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala
1 5 10
<210> 23
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Trp Ala Ser Thr Arg His Thr
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gln Gln His Tyr Asn Thr Pro Leu Thr
1 5
<210> 25
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val His Ile Ser Cys Lys Ala Asn Gly Tyr Thr Tyr Ser Asp Tyr
20 25 30
Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Leu Tyr Gly Asn Gly Arg Thr Arg Tyr Asn Glu Lys Leu
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Ala Ser Tyr Gly Asp Tyr Ala Asp Tyr Trp Gly His Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 26
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105