阅读说明：本技术 T细胞抗原受体、其多聚体复合物及其制备方法和应用 (T cell antigen receptor, polymer compound thereof, preparation method and application thereof ) 是由 陈花 刘光娜 雷蕾 刘芳 贾乐梅 黄道盛 赵学强 林欣 于 2021-03-04 设计创作，主要内容包括：本发明提供了一种T细胞抗原受体、表达T细胞抗原受体(TCR)的免疫细胞,及其制备方法和应用。本发明所述的TCR能够特异性地被病毒抗原肽提呈细胞激活,提高胞外细胞因子IFNγ、IL2的释放水平及乳酸脱氢酶释放量,且显著地杀伤靶细胞。(The invention provides a T cell antigen receptor, an immune cell expressing the T cell antigen receptor (TCR), a preparation method and application thereof. The TCR can be specifically activated by virus antigen peptide presenting cells, improves the release level of extracellular cytokines IFN gamma and IL2 and the release amount of lactate dehydrogenase, and obviously kills target cells.)

1. A T cell antigen receptor that specifically binds CMV pp65, comprising the CDRs 1 a-CDR 3 a of the α chain and the CDRs 1 β -CDR3 β of the β chain, wherein the amino acid sequence of CDR1 a comprises SEQ ID NO: 1-2 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 1-2, and the amino acid sequence of CDR2 α comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-4 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 3-4, and the amino acid sequence of CDR3 a comprises the amino acid sequence set forth in SEQ ID NO: 5-6 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 5-6, and the amino acid sequence of CDR1 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-8 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 7-8, and the amino acid sequence of CDR2 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-10 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 9-10, and the amino acid sequence of CDR3 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-12 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11-12 has at least 80% homology;

preferably, the CDRs 1 α -CDR3 α and 1 β -CDR3 β comprise any one of the following groups:

2. the T-cell antigen receptor according to claim 1, characterized in that the amino acid sequence of its beta chain comprises SEQ ID NO: 34-35 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 34-35, and the amino acid sequence of the alpha chain comprises SEQ ID NO: 36-37 or a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 36-37 has at least 80% homology with the amino acid sequence shown in any one of the amino acid sequences;

preferably, the α chain is linked directly or indirectly, preferably indirectly, to the amino acids of the β chain, more preferably via fp 2A;

further preferably, the amino acid sequence of the T cell antigen receptor comprises SEQ ID NO: 30 or 32 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 30 or 32 has at least 80% homology.

3. A nucleic acid encoding the T cell antigen receptor of any one of claims 1-2.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell or immune cell comprising the nucleic acid of claim 3 or the expression vector of claim 4, wherein the immune cell expresses the T cell antigen receptor of any one of claims 1-2, preferably wherein the immune cell is selected from the group consisting of a T cell and a stem cell, and further preferably wherein the immune cell is isolated from a T cell derived from a subject.

6. A method for producing a recombinant T cell, comprising the steps of:

1) cloning a nucleic acid according to claim 3 from a positive T cell;

2) separating and culturing primary T cells;

3) delivering the nucleic acid obtained in step 1) to the primary T cell described in step 2) to obtain a recombinant T cell expressing the T cell antigen receptor of any one of claims 1-2;

preferably, the method of making further comprises the step of knocking out an endogenous TCR in the cell.

7. A method for preparing a T cell antigen receptor, comprising the steps of:

(1) cloning a nucleic acid according to claim 3 from a positive T cell;

(2) connecting the nucleic acid obtained in the step (1) to a vector skeleton to obtain an expression vector;

(3) transforming the expression vector obtained in the step (2) into a host cell, and then inducing the expression of the host cell;

(4) obtaining the T cell antigen receptor.

8. A multimeric complex, comprising a T-cell antigen receptor according to any one of claims 1-2.

9. The multimeric complex of claim 8, further comprising a monomer, a biotin molecule, and a streptavidin or avidin molecule, wherein the monomer comprises an extracellular domain of an MHC molecule a chain and a β 2m chain and an antigenic peptide, the monomer is conjugated to the biotin molecule, and the biotin molecule is conjugated to the streptavidin or avidin molecule;

preferably, the antigenic peptide comprises SEQ ID NO: 13,

preferably, the MHC molecule is HLA-a 1101.

10. A method of preparing a multimeric complex according to any one of claims 8 to 9, comprising the steps of:

I) expressing and purifying an MHC molecule alpha chain extracellular region and a beta 2m chain which are connected with an avi-tag sequence at the C terminal;

II) carrying out refolding on the antigen peptide, the beta 2m chain obtained in the step I) and an MHC molecule alpha chain extracellular region connected with an avi-tag sequence at the C end to prepare a monomer;

III) biotinylating the monomer prepared in the step II) to obtain a biotinylated monomer;

IV) reacting the biotinylated monomer obtained in the step III) with streptavidin or avidin with fluorescent markers to prepare an antigen peptide-MHC molecule tetramer;

v) incubating the antigen peptide-MHC molecule tetramer obtained in the step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer complex.

11. Use of the T-cell antigen receptor of any one of claims 1-2, the nucleic acid of claim 3, the expression vector of claim 4, the host cell of claim 5, the immune cell of claim 5, the multimeric complex of any one of claims 8-9 for the preparation of a product for the diagnosis or treatment of a tumor or a disease associated with CMV or for T-cell labeling, detection, cell sorting or activation.

12. A pharmaceutical composition or kit comprising any one of the following groups:

i) the T cell antigen receptor of any one of claims 1-2;

ii) the nucleic acid of claim 3;

iii) the expression vector of claim 4;

iv) the host cell of claim 5;

v) the immune cell of claim 5; or

vi) the multimeric complex of any one of claims 8-9.

Technical Field

The present invention relates to the field of T cell antigen receptor technology capable of recognising CMV pp65, in particular to various TCRs and their use in the treatment and/or prevention of CMV-related diseases.

Background

The T cell antigen receptor (TCR) is crucial to the cellular immune function of the immune system, and the TCR is the only receptor of specific antigen peptide presented on Major Histocompatibility Complex (MHC), and the combination of the antigen specific TCR and the pMHC complex triggers the direct physical contact and interaction of the T cell and the antigen presenting cell, leads to a series of subsequent cell signal transmission and other physiological reactions, thereby enabling the T cells with different antigen specificities to exert immune effects on target cells thereof.

CN107827959A discloses a method for isolating antigen-specific T cells and cloning antigen-specific TCR-encoding genes in antigen-specific T cells. The TCR can be combined with HBVS183-91 antigen short peptide complex FLLTRILTI-HLA A0201, and cells transduced with the TCR can be specifically activated and have strong killing effect on target cells. CN106279404A discloses a soluble and stable heterodimeric TCR comprising an artificial interchain disulfide bond between the α chain variable region and the β chain constant region, which is soluble and stable, can be renatured, refolded, purified well and at the same time can bind specifically to the protoligand. None of the above patents, however, discloses a specific TCR against cytomegalovirus.

Cytomegalovirus (CMV) is a herpesvirus DNA virus, also known as a cell inclusion virus. The cytomegalovirus is widely distributed, the infection is very common in people, the infection rate of Chinese adults reaches more than 95 percent, negative infection is usually caused, most infected people have no clinical symptoms, and inflammatory diseases can be generated by attacking a plurality of organs and systems under certain conditions. CN102656188A discloses a T cell receptor capable of recognizing antigens from cytomegalovirus, which specifically binds to a peptide from the Cytomegalovirus (CMV) phosphoprotein pp65 having the amino acid sequence NLVPMVATV when presented by the Major Histocompatibility Complex (MHC). However, the killing effect of the T cell receptor on the target cell is not significant in this application.

Therefore, the invention provides a T cell antigen receptor which can be specifically activated by virus antigen peptide presenting cells, improve the release level of extracellular cytokines IFN gamma and IL2 and the release amount of lactate dehydrogenase, and remarkably kill target cells.

Disclosure of Invention

The present invention provides a plurality of T cell antigen receptors (TCRs) that specifically bind Cytomegalovirus (CMV) phosphoprotein pp65 antigenic peptides presented by MHC molecules, said Cytomegalovirus (CMV) phosphoprotein pp65 comprising the amino acid sequence of SEQ ID NO: 28, and the nucleotide sequence is shown as SEQ ID NO: as shown at 29. The TCR can specifically recognize a corresponding CMV pp65 antigen peptide-MHC molecule compound, activate TCR-T cells, further generate high-level cytokines IFN gamma, IL2 and TNF alpha, and remarkably kill tumor cells in vivo and in vitro tests. The method comprises the following specific steps:

in a first aspect of the invention, there is provided a T cell antigen receptor which specifically binds CMV pp 65. Preferably, the binding epitope comprises SEQ ID NO: 13.

preferably, the T cell antigen receptor specifically binds to an antigenic peptide from the Cytomegalovirus (CMV) phosphoprotein pp65 by presentation on Major Histocompatibility Complex (MHC).

Preferably, the T cell antigen receptor comprises at least one alpha chain variable region and/or one beta chain variable region.

Preferably, the T cell antigen receptor is an α β heterodimer.

Preferably, the T cell antigen receptor comprises the alpha chain CDRs 1 alpha-CDR 3 alpha and the beta chain CDRs 1 beta-CDR 3 beta.

The amino acid sequence of CDR1 α comprises SEQ ID NO: 1-2 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 1-2, and the amino acid sequence of CDR2 α comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-4 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 3-4, and the amino acid sequence of CDR3 a comprises the amino acid sequence set forth in SEQ ID NO: 5-6 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 5-6, and the amino acid sequence of CDR1 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-8 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 7-8, and the amino acid sequence of CDR2 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-10 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 9-10, and the amino acid sequence of CDR3 β comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-12 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11-12 has at least 80% homology;

in one embodiment of the invention, the CDRs 1 α -CDR3 α and 1 β -CDR3 β comprise any one of the following groups:

in one embodiment of the invention, the amino acid sequence of its beta strand comprises SEQ ID NO: 34-35 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 34-35 has at least 80% homology.

In one embodiment of the invention, the amino acid sequence of the α chain comprises SEQ ID NO: 36-37 or a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 36-37 has at least 80% homology.

Preferably, the alpha chain is linked directly or indirectly, preferably indirectly, to the amino acids of the beta chain, more preferably via fp2A (furin-SGSG-p 2A).

Further preferably, the amino acid sequence of fp2A comprises SEQ ID NO: 215.

in one embodiment of the present invention, the connection sequence of the α chain and the β chain may be α chain, fp2A and β chain, or β chain, fp2A and α chain.

In one embodiment of the present invention, it is further preferred that the amino acid sequence of the T cell antigen receptor comprises SEQ ID NO: 30 or 32 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 30 or 32 has at least 80% homology.

In a second aspect of the invention, an antibody or antigen-binding fragment thereof is provided that specifically binds CMV pp 65. Preferably, the binding epitope comprises SEQ ID NO: 13.

preferably, the antibody or antigen binding fragment thereof comprises the α chain CDR1 α -CDR3 α and the β chain CDR1 β -CDR3 β.

In one embodiment of the invention, the amino acid sequence of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30 or 32 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 30 or 32 has at least 80% homology.

Preferably, the antibody or antigen binding fragment thereof may further comprise a fragment such as a Fab, Fab '-SH, Fv, scFv, (Fab') 2, single domain antibody, diabody (dAb), or linear antibody.

In a third aspect of the invention, there is provided a nucleic acid encoding the beta chain of the T cell antigen receptor of the invention.

Preferably, the nucleic acid sequence encoding the β chain of the T cell antigen receptor comprises SEQ ID NO: 38 or 39 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 38 or 39 has at least 80% homology.

In a fourth aspect of the invention, there is provided a nucleic acid encoding the alpha chain of the T cell antigen receptor of the invention.

Preferably, the nucleic acid sequence encoding the alpha chain of the T cell antigen receptor comprises SEQ ID NO: 40 or 41 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 40 or 41 has at least 80% homology.

In a fifth aspect of the invention, there is provided a nucleic acid encoding a T cell antigen receptor or antibody or antigen binding fragment thereof according to the invention.

Preferably, the alpha chain and the beta chain of the T cell antigen receptor are connected by a nucleic acid sequence coding fp 2A. Further preferably, said nucleic acid sequence encoding fp2A comprises SEQ ID NO: 216.

preferably, the nucleic acid sequence encoding the T cell antigen receptor comprises SEQ ID NO: 31 or 33, or comprises a sequence identical to SEQ ID NO: 31 or 33 has at least 80% homology.

Preferably, the nucleic acid sequence encoding the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 31 or 33, or comprises a sequence identical to SEQ ID NO: 31 or 33 has at least 80% homology.

The nucleic acid sequence of the present invention may be single-stranded or double-stranded, and may be DNA or RNA.

Preferably, the nucleic acid sequence may be codon optimized. Further preferably, said codon optimisation comprises changing a number of rare codons used by the virus etc. to corresponding mammalian codons and/or removing mRNA instability motifs and/or cryptic splice sites.

In a sixth aspect of the invention, there is provided an expression vector comprising any one of the nucleic acids of the invention.

Preferably, the expression vector is capable of expression in vivo or in vitro or ex vivo. Further preferably, the expression vector is expressed at a high level in vivo in cells.

Preferably, the expression vector may be a prokaryotic expression vector or a retroviral vector.

Further preferably, the prokaryotic expression vector is an escherichia coli series. In one embodiment of the invention, the expression vector is pET-26b or pET28a +.

Further preferably, the virus may be Rous Sarcoma Virus (RSV), lentivirus, Human Immunodeficiency Virus (HIV), Murine Leukemia Virus (MLV), Equine Infectious Anemia Virus (EIAV), mouse mammary carcinoma virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine sarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloproliferative virus 29(MC29), avian myeloblastosis virus (AEV), or the like. Still further preferably, the expression vector is a lentiviral expression vector.

In one embodiment of the invention, the expression vector is pHAGE-IRES-RFP.

Further preferably, the connection sequence of the beta chain and the alpha chain in the expression vector and the skeleton vector is a promoter, the beta chain, fp2A, the alpha chain, IRES and RFP sequence.

In a seventh aspect of the invention, there is provided a host cell comprising a nucleic acid or expression vector according to any one of the invention.

Preferably, the host cell may be eukaryotic or prokaryotic. More preferably, the host cell is a yeast cell, 293 cell, CHO cell, E.coli, etc.

In a specific embodiment of the present invention, the host cell is Stbl3, BL21 or transetta.

In an eighth aspect of the invention, there is provided an immune cell expressing a T cell antigen receptor or antibody or antigen binding fragment thereof according to the invention.

Preferably, the immune cell comprises one or more heterologous nucleic acid sequences according to any of the present invention.

Preferably, the immune cells include, but are not limited to, stem cells, lymphocytes (including T cells, B cells). Further, the immune cell is a B cell that expresses the antibody or antigen-binding fragment thereof described above. The immune cell is a T cell whose T cell antigen receptor structure is as defined above.

Preferably, the T cell may be CD4+ T, CD8+ T, etc.

Further preferably, the immune cells are derived from T cells isolated from the subject.

Preferably, the immune cell is a T cell from a CMV seronegative donor.

In a ninth aspect of the present invention, there is provided a method for preparing an immune cell, comprising transfecting a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof, or the T cell antigen receptor into an immune cell and expressing the nucleic acid sequence.

Preferably, the immune cells include, but are not limited to, stem cells, lymphocytes (including T cells, B cells). Further, the immune cell is a B cell that expresses the antibody or antigen-binding fragment thereof described above. The immune cell is a T cell whose T cell antigen receptor structure is as defined above.

Preferably, the method further comprises the step of knocking out an endogenous TCR of the cell. Specifically, the guide targeting the endogenous TCR can be constructed into a lentiviral vector, co-transformed with a packaging plasmid, a transfection reagent, to a T cell.

In a tenth aspect of the present invention, there is provided a method for preparing a recombinant T cell, comprising the steps of:

1) obtaining a nucleic acid according to any of the present invention from a positive T cell clone;

2) separating and culturing primary T cells;

3) delivering the nucleic acid obtained in the step 1) to the primary T cell in the step 2) to obtain the recombinant T cell expressing any T cell antigen receptor of the invention.

Preferably, the T cells are selected from hematopoietic stem cells or Peripheral Blood Lymphocyte (PBL) -derived T cells.

In an eleventh aspect of the invention, there is provided a method of producing an antibody or an antigen-binding fragment thereof or a T cell antigen receptor, comprising the steps of:

(1) obtaining a nucleic acid according to any of the present invention from a positive T cell clone;

(2) connecting the nucleic acid obtained in the step (1) to a vector skeleton to obtain an expression vector;

(3) transforming the expression vector obtained in the step (2) into a host cell, and then inducing the expression of the host cell;

(4) obtaining the antibody or antigen binding fragment thereof or the T cell antigen receptor.

Preferably, the positive T cell is specifically bound with an MHC presented Cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide. Further preferably, the MHC presented Cytomegalovirus (CMV) phosphoprotein pp65 antigenic peptide complex is a monomer or a multimer complex.

In a twelfth aspect of the invention, there is provided a multimeric complex comprising a T cell antigen receptor according to any one of the invention.

Preferably, the multimeric complex further comprises a monomer, a biotin molecule, and a streptavidin or avidin molecule, wherein the monomer comprises an MHC molecule extracellular domain and a β 2m chain and an antigenic peptide, the monomer is conjugated to the biotin molecule, and the biotin molecule is bound to the streptavidin or avidin molecule.

Preferably, the MHC molecule alpha chain extracellular region is connected with an avi-tag sequence at the C terminal.

Preferably, the extracellular domain of the MHC molecule alpha chain does not contain a signal peptide sequence. And the M amino acid is added in front of the mature peptide sequence.

Preferably, the β 2m chain does not contain a signal peptide sequence. And two amino acids, M and A, are added in front of the mature peptide sequence.

In one embodiment of the invention, the β 2m chain is free of signal peptide and is preceded by two amino acids, preferably M, A, before the mature peptide sequence.

Preferably, the antigenic peptide comprises SEQ ID NO: 13.

in one embodiment of the invention, the multimeric complex comprises:

(1) a T cell antigen receptor; preferably comprises SEQ ID NO: 30 or 32 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 30 or 32 has at least 80% homology.

(3) The monomer comprises an antigen peptide, an MHC molecule alpha chain extracellular region connected with an avi-tag sequence at the C terminal, and a beta 2m chain without a signal peptide; the antigenic peptide is SEQ ID NO: 13;

(4) a biotin molecule; and

(5) a streptavidin molecule or an avidin molecule; wherein the monomer is conjugated to the biotin molecule which binds to the streptavidin or avidin.

Preferably, the MHC molecule is an MHC class I molecule or an MHC class II molecule. More preferably, the MHC molecule is an MHC class I molecule.

Preferably, the MHC molecule is HLA-a 1101.

In one embodiment of the present invention, the amino acid sequence of the α chain of the MHC molecule is as shown in SEQ ID NO: 24 or SEQ ID NO: 24 has at least 80% homology.

In one embodiment of the present invention, the nucleotide sequence of the MHC molecule α chain is as shown in SEQ ID NO: 25 or SEQ ID NO: 25 has at least 80% homology.

In one embodiment of the invention, the amino acid sequence of the β 2m chain of the MHC molecule is as set forth in SEQ ID NO: 26 or SEQ ID NO: 26 has at least 80% homology.

In one embodiment of the invention, the nucleotide sequence of the β 2m chain of the MHC molecule is as set forth in SEQ ID NO: 27 or SEQ ID NO: 27 has at least 80% homology.

To increase the specificity of binding of the antigenic peptide-MHC tetramer to a T cell antigen receptor, the monomer further comprises a chemical modification, mutation, insertion and/or deletion of at least one amino acid.

Preferably, the extracellular domain of the alpha chain and the beta 2m chain of the MHC molecule are non-covalently bound.

Preferably, the multimeric complex comprises at least one monomer.

Preferably, at least one biotin molecule is coupled per monomer.

In a thirteenth aspect of the invention, there is provided a method of preparing a multimeric complex according to any one of the invention, comprising the steps of:

I) expressing and purifying an MHC molecule alpha chain extracellular region and a beta 2m chain which are connected with an avi-tag sequence at the C terminal;

II) carrying out refolding on the antigen peptide, the beta 2m chain obtained in the step I) and an MHC molecule alpha chain extracellular region connected with an avi-tag sequence at the C end to prepare a monomer;

III) biotinylating the monomer prepared in the step II) to obtain a biotinylated monomer;

IV) reacting the biotinylated monomer obtained in the step III) with streptavidin or avidin with fluorescent markers to prepare an antigen peptide-MHC molecule tetramer;

v) incubating the antigen peptide-MHC molecule tetramer obtained in the step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer complex.

Preferably, the step I) includes cloning a nucleotide sequence of an extracellular domain of an MHC molecule α chain encoding a C-terminal-linked avi-tag sequence and a nucleotide sequence of an MHC molecule β 2m chain, respectively, linking the cloned nucleotide sequences to a vector, transforming the vector into an expression strain, culturing the vector, adding an inducer, and extracting the inclusion body.

Further preferably, the expression strain is cultured to OD₆₀₀The value is between 0.2 and 0.4.

Further preferably, the final molarity of the inducer after addition is between 0.5 and 1 mM.

Preferably, the expression is induced for 4-6 h.

Preferably, the step II) includes refolding β 2m chain, that is, adding antigenic peptide, MHC molecule β 2m chain, and MHC molecule α chain extracellular region with C-terminal connected to avi-tag sequence into dilution buffer in sequence in a water bath protected from light, wherein refolding β 2m chain includes denaturation of inclusion body, addition of protease inhibitor, and dialysis.

Preferably, the molar ratio of the antigenic peptide, the beta 2m chain without the signal peptide and the alpha chain of the C-terminal connecting avi-tag sequence is (30-50): (2-2.5): 1. more preferably 40: 2:1.

Preferably, the step II) further comprises a step of purifying the monomer.

Preferably, the biotinylation in step III) is carried out by binding the monomer to BiomixA and BiomixB under the catalysis of BirA enzyme.

Preferably, said step III) further comprises a step of purifying the biotinylated monomer.

Preferably, the molar ratio of the monomer reacted with streptavidin in the step IV) is (4-7): 1.

in a fourteenth aspect of the present invention, there is provided a use of the above multimeric complex for preparing, screening or detecting the antibody or antigen-binding fragment thereof or T cell antigen receptor of the present invention.

In a fifteenth aspect of the present invention, there is provided a method for preparing a T cell antigen receptor, comprising the steps of:

I) expressing and purifying an MHC molecule alpha chain extracellular region and a beta 2m chain which are connected with an avi-tag sequence at the C terminal;

II) folding the antigen peptide, the MHC molecule alpha chain extracellular region connected with the avi-tag sequence at the C end and obtained in the step I) and the beta 2m chain in a renaturation way to prepare a monomer;

III) biotinylating the monomer prepared in the step II) to obtain a biotinylated monomer;

IV) reacting the biotinylated monomer obtained in the step III) with streptavidin or avidin with fluorescent markers to prepare an antigen peptide-MHC molecule tetramer;

v) incubating the antigen peptide-MHC molecule tetramer obtained in the step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer compound, and separating to obtain the T cell antigen receptor.

In a sixteenth aspect of the present invention, there is provided a use of any one of the T cell antigen receptors of the present invention, any one of the antibodies or antigen binding fragments thereof of the present invention, any one of the nucleic acids of the present invention, the expression vector of the present invention, the host cell of the present invention, the immune cell of the present invention, or any one of the multimeric complexes of the present invention in the preparation of a product for diagnosing or treating a tumor or a disease associated with CMV.

Preferably, the CMV-associated disease is selected from the group consisting of neonatal CMV inclusion body disease, acute acquired CMV infection, or a disease caused by infection with CMV in an immunocompromised person.

In one embodiment of the invention, the CMV-associated disease is selected from liver, spleen or central nervous system diseases, disabilities, infectious mononucleosis, skeletal muscle pain, CMV retinitis, gastrointestinal CMV or encephalitis, and the like.

In a seventeenth aspect of the invention, there is provided a T cell antigen receptor according to any one of the present invention, an antibody or antigen binding fragment thereof according to any one of the present invention, a nucleic acid according to any one of the present invention, an expression vector according to the present invention, a host cell according to the present invention, an immune cell according to the present invention, or a multimeric complex according to any one of the present invention for use in T cell labelling, detection, cell sorting or activation.

In an eighteenth aspect of the present invention, there is provided a pharmaceutical composition comprising any one of the following groups:

i) the T cell antigen receptor of the invention;

ii) a nucleic acid according to the invention;

iii) an expression vector according to the invention;

iv) a host cell according to the invention;

v) an immune cell according to the invention;

vi) a multimeric complex according to the invention; or

vii) an antibody or antigen-binding fragment thereof according to the invention.

Preferably, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition may also be used in combination with other therapeutic agents. Further preferably, the therapeutic agent may be an immunomodulator.

In a nineteenth aspect of the present invention, there is provided a kit comprising any one of the following groups:

i) the T cell antigen receptor of the invention;

ii) a nucleic acid according to the invention;

iii) an expression vector according to the invention;

iv) a host cell according to the invention;

v) an immune cell according to the invention;

vi) a multimeric complex according to the invention; or

vii) an antibody or antigen-binding fragment thereof according to the invention.

In a twentieth aspect of the invention, there is provided a method for detecting Cytomegalovirus (CMV) phosphoprotein pp65, comprising contacting a sample to be tested with an antibody or antigen-binding fragment thereof or a T-cell antigen receptor of the invention, and detecting a complex formed between Cytomegalovirus (CMV) phosphoprotein pp65 and the antibody or antigen-binding fragment thereof or the T-cell antigen receptor.

Preferably, the detection of Cytomegalovirus (CMV) phosphoprotein pp65 is the detection of the presence or amount of Cytomegalovirus (CMV) phosphoprotein pp 65. Wherein the presence or absence is indicated, and the content may be an expression amount, a protein concentration, or the like.

Preferably, the antibody or antigen-binding fragment thereof or T cell antigen receptor comprises a detectable label.

In one embodiment of the invention, the tag may be His and/or HA.

The method for detecting Cytomegalovirus (CMV) phosphoprotein pp65 is not a disease diagnosis method. Firstly, the sample to be examined is not an organism or an ex vivo tissue or cell thereof, and secondly, even the presence of the Cytomegalovirus (CMV) phosphoprotein pp65 in an organism or the inclusion of a concentration or expression level of the Cytomegalovirus (CMV) phosphoprotein pp65 is not a definite disease, but a possibility.

In a twenty-first aspect of the present invention, there is provided a method of treating and/or preventing a disease associated with CMV comprising administering to an individual an effective amount of the antibody or antigen-binding fragment thereof, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell, or the pharmaceutical composition of the invention.

Preferably, the method comprises the step of adoptively transferring T cells expressing the T cell antigen receptor of the invention to the subject.

Preferably, the method comprises locating a T cell antigen receptor of the invention in the vicinity of a disease associated with CMV, preferably a tumor or a metastatic tumor, to increase the efficacy of the toxin or the immunostimulant.

Preferably, for use in the treatment and/or prevention of CMV reactivation following allogeneic hematopoietic stem cell transplantation.

Preferably, it is used for the treatment and/or prevention of reactivation of CMV following organ transplantation (e.g. kidney, liver, pancreas, intestine, cornea), tissue transplantation, cell transplantation (islet cells, limbal stem cells) or stem cell therapy.

Preferably, said T cell expressing said T cell antigen receptor of the invention is derived from a subject.

Preferably, said T cell expressing said T cell antigen receptor of the invention is derived from the same donor as said hematopoietic stem cell, organ, tissue, cell or stem cell.

In a twenty-second aspect of the invention, there is provided a method of diagnosing a disease associated with CMV comprising taking a sample, contacting the sample with an antibody or antigen-binding fragment thereof or a T-cell antigen receptor of the invention and detecting a complex formed by CMV pp65 with the antibody or antigen-binding fragment thereof or the T-cell antigen receptor.

Preferably, the antibody or antigen-binding fragment thereof or T cell antigen receptor comprises a detectable label.

The TCR can specifically recognize a corresponding CMV pp65 antigen peptide-MHC molecule compound, activate TCR-T cells, further generate high-level cytokines IFN gamma, IL2 and TNF alpha, and remarkably kill tumor cells in vivo and in vitro tests.

The "T cell antigen receptor" according to the present invention is a molecule capable of recognizing a peptide when presented by an MHC molecule or its tetramer.

The "tumor" according to the present invention includes, but is not limited to, pancreatic cancer, liver cancer, colon cancer, rectal cancer, stomach cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. Preferably, the tumor is selected from pancreatic cancer, liver cancer, oral squamous carcinoma, colon cancer, ovarian cancer, and gastric cancer. In one embodiment of the invention, the tumor is a lymphoma.

The "CMV-associated disease" described herein includes diseases caused by infection with CMV pp65, including neonatal CMV inclusion body disease, which can seriously affect the liver, spleen and central nervous system, as well as disability. Also included are acute acquired CMV infections, similar to infectious mononucleosis, including fever, skeletal muscle pain, and the like. Also included are immunocompromised persons, such as organ-transplanted persons, or persons with HIV, infections that can lead to CMV retinitis, gastrointestinal CMV and encephalitis, and the like.

As used herein to describe a sequence of a protein or nucleic acid, the "comprising" of the invention may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described herein.

"prevention" as referred to herein means suppression of symptoms or delay of all actions of a particular symptom stress by administration of a product as described herein.

"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.

"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

An "effective amount" as referred to herein refers to an amount or dose of a product of the invention which provides the desired treatment or prevention after administration to a patient or organ in single or multiple doses.

The "product" of the present invention includes, but is not limited to, the antibody or antigen-binding fragment thereof, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell or the multimeric complex of the present invention, and other agents that assist or cooperate with the above-mentioned products.

The product of the invention can be a kit, a chip, an antibody conjugate or a multifunctional antibody and other pharmaceutical compositions.

The "subject" of the present invention includes, but is not limited to, a human or non-human mammal. Preferably, the non-human mammal includes, but is not limited to, mouse, rat, monkey, pig or rabbit.

"homology" as used herein means that in the context of using an amino acid sequence or a nucleotide sequence, one skilled in the art can adjust the sequence according to the actual working requirement without changing the main structure or function of the original sequence, such that the used sequence has (including but not limited to) 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology. For example, the term "a sequence identical to SEQ ID NO: 1-2 "is at least 80% homologous" to the amino acid sequence set forth in any one of claims 1-2, in that the amino acid sequence retains the peptide epitope with Cytomegalovirus (CMV) phosphoprotein pp 65: on the premise of the MHC complex binding function, the amino acid sequence of SEQ ID NO: 1 or 2, comprising one or more alterations such as substitutions, deletions and/or insertions of one or more amino acids, etc., truncations, or one or both ends lengthened, as long as they retain more than 80% homology and retain the epitope with pp 65: MHC complex or pp65 epitope: the ability of an MHC molecule to tetramer bind. The alteration may be a substitution, an addition or a deletion. May be a substitution between amino acids of the same polarity, a substitution between amino acids of the same charge, a substitution between amino acids having no charge, a substitution between aliphatic amino acids, a substitution between aromatic amino acids, a substitution between amino acids having no polarity, or a substitution between amino acids having a side chain moiety of a relevant property. Among these, basic side chains include, but are not limited to, lysine, arginine, or histidine. Acidic side chains include, but are not limited to, aspartic acid or glutamic acid. Uncharged amino acids include, but are not limited to, aspartyl, glutamine, serine, threonine, or tyrosine. Nonpolar side chains include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, or cysteine. Wherein said at least 80% includes but is not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: SDS PAGE detection result of the antigen peptide-MHC monomer, wherein M is a protein Marker, and the strips 1 and 2 are monomers;

FIG. 2: A1101-ATV tetramer detects the positive rate of C44-TCR-T cells;

FIG. 3: the connection of the TCR beta chain and the alpha chain in a pHAGE vector is schematically shown, wherein the connection sequence is a promoter, the beta chain, furin-p2A, the alpha chain, an IRES and an RFP sequence in sequence;

FIG. 4: detecting the membrane surface expression of the TCR by flow cytometry, in particular the membrane surface expression of HLA-A1101 ATVQGQNLK specific TCR (C44, C45), wherein BV421 is one of serial luciferins and BV is called Brilliant Violet;

FIG. 5: flow cytometry detects the binding affinity of the TCR and the CMV pp65 tetramer probe, in particular the binding affinity detection result of HLA-A1101 ATVQGQNLK specific TCR (C44, C45) and the CMV pp65 tetramer probe;

FIG. 6: a graph showing the results of measurements of the release level of C44-TCR-T, C45-TCR-T cytokine IFN γ and the luciferase level of target cells, wherein C44 and C45 are TCRs prepared in example 2, NE represents a blank control group, NT represents a T cell-only group, Raji is an Raji cell untransformed pp65, and 1G4 represents a TCR specific for NY-ESO-1;

FIG. 7: C45-TCR was evaluated for inhibition of tumor growth in mice in a lymphoma animal model;

FIG. 8: lymphoma animal model, statistics of tumor growth in mice without T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C45-TCR);

FIG. 9: lymphoma animal model, statistics of TCR-T cell specific proliferation in mice without T cell injection group (PBS), control T cell injection group (NC), and CMV TCR-T injection group (C45-TCR).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: construction and effect detection of CMV epitope tetramer

Construction of CMV epitope tetramer

1) Expression sequence optimized HLA-a 1101 (amino acid sequence shown in SEQ ID NO: 24, and the nucleotide sequence is shown as SEQ ID NO: 25) and a beta 2m chain (the amino acid sequence of which is shown in SEQ ID NO: 26, and the nucleotide sequence is shown as SEQ ID NO: shown at 27). Wherein the structure of the alpha chain is that the extracellular region sequence of the corresponding HLA type alpha chain is linked with an Avi-tag sequence, and the Avi-tag sequence is separated by a BamHI enzyme cutting site to provide a biotinylation site. The β 2M chain removes the signal peptide sequence and adds two amino acids (M and A) in front of the mature peptide sequence. The expression vector is PET28a +, and the expression bacteria are transetta or BL 21. IPTG concentration was 0.5mM, and expression was induced for 4 h. Extracting alpha chain and beta 2m chain protein inclusion bodies.

2) Selection of CMV epitopes (antigenic peptides): HLA-A1101 type corresponds to epitope ATVQGQNLK (SEQ ID NO: 13).

3) pMHC I monomer folding and purification: adding the antigenic peptide obtained in the step 2), the beta 2m chain renaturation protein obtained in the step 1) and the alpha chain protein into a reduction system in sequence according to the molar ratio of 40:2:1, and carrying out folding reaction for 72 h. The resulting product was purified on a Superdex 7510/300 GL column. And collecting the purified product, biotinylating the purified product by using an avidity kit, purifying again to obtain a biotinylated monomer, and detecting the purity of the monomer by gel electrophoresis.

4) And (3) carrying out binding reaction on the biotinylated monomer in the step 3) and the APC-labeled streptavidin to obtain a corresponding tetramer, namely A1101-ATVQGQNLK-tetramer (A1101-ATV tetramer for short).

Detection of CMV epitope tetramer effect

1. Isolating human Peripheral Blood Mononuclear Cells (PBMC) or preparing TCR-T cells and preparing a cell suspension at a cell density of 1X 10⁶cells/mL.

2. Cells were centrifuged at 3000rpm for 5 min. The supernatant was removed and resuspended in 50. mu.L of PBS containing 1% serum.

3. Add 2. mu.L of tetramer and incubate at room temperature for 30 min.

4. Add 2. mu.L of CD8 antibody and incubate for 20min on ice.

5. 1mL PBS was added at 3000rpm for 5 min.

6. The supernatant was removed and 1mL PBS was added at 3000rpm for 5 min.

7. The supernatant was removed, the cells were resuspended in 500. mu.L of 4% paraformaldehyde, and the cell suspension was filtered through a filter.

8. Positive cells were detected by flow cytometry.

Third, experimental results

The SDS PAGE of the monomers was determined as shown in FIG. 1. As shown in FIG. 1, the resulting monomer clearly shows proteins of corresponding sizes of heavy chain (alpha chain extracellular region with C terminal connected with Avi-tag sequence) and light chain (beta 2m chain with signal peptide region removed) through renaturation folding and HPLC purification, and has high purity.

And respectively incubating the constructed tetramer with corresponding HLA type PBMC cells to obtain specific T cells. The corresponding TCR of type a1101 (C44-TCR) was recognized by the same epitope as the self-developed and commercial tetramer (MBL, TS-M012-1) (see fig. 2), with the commercial tetramer-positive population accounting for 90.5% and the self-developed tetramer-positive population accounting for 91% of the infected cells. C45-TCR results are shown in FIG. 4.

Example 2: construction of pHAGE-TCR-RFP vector

First, get the beta and alpha gene segments of the TCR with CMV pp65 epitope specificity

1) Using HLA-A1101-ATVQGQNLK tetramer purchased from MBL company or self-made by the company, staining peripheral blood according to the product instruction, subjecting T cells positive in tetramer staining to flow single cell sorting, and reverse transcription to obtain cDNA: (IV Reverse Transcriptase, Invitrogen). The variable region fragment of the TCR β gene was amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO) according to the multiplex PCR (Multiplex PCR) principle.

The reverse transcription primer is as follows: TRBC1-TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 136)

The PCR amplification primers are as follows:

an upstream primer 1: t cell receptor beta variable region- -TRBV- -F1 (SEQ ID NOS: 56 to 95, wherein SEQ ID NOS: 60, 59 can also be used alone as TRBV- -F1 for preparing variable region fragments of the beta genes of C44, C45, respectively)

An upstream primer 2: t cell receptor beta variable region- -TRBV- -F2 (SEQ ID NOS: 96 to 135, wherein SEQ ID NOS: 102, 100 can also be used individually as TRBV- -F2 for preparing variable region fragments of the beta genes of C44, C45, respectively)

A downstream primer 1: t cell receptor beta constant region- -TRBC2-GCACCTCCTTCCCATTCACC (SEQ ID NO: 137)

A downstream primer 2: t cell receptor beta constant region-TRBC 3-GCTTCTGATGGCTCAAACACAG (SEQ ID NO: 138)

Specifically, according to the instructions of the PCR polymerase KOD-Plus-Neo product, the PCR system is 20 μ L in one round, the annealing temperature is 60 ℃, and the reaction is carried out for 30 cycles. Taking 1 mu L of the product of the first round of PCR reaction as a template of the second round of PCR, wherein the second round of PCR system is 30 mu L, the annealing temperature is 60 ℃, and the reaction is carried out for 30 cycles. And then, running agarose gel on the second round PCR product, cutting and recovering the bands with corresponding sizes (Tiangen gel recovery kit), and sequencing, wherein the sequencing primer is a downstream primer 2. Obtaining the TCR beta gene sequence. The beta chain nucleotide sequences of C44 and C45 are shown as SEQ ID NO: 38. shown at 39.

2) As above, cDNA was obtained by reverse transcription of T cells positive for tetramer staining: (IV Reverse Transcriptase, Invitrogen). TCR alpha gene fragments were amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO) according to the product instructions.

The reverse transcription primer is as follows: t cell receptor alpha constant region-TRAC 1-CGACCAGCTTGACATCACAG (SEQ ID NO: 139)

The PCR amplification primers are as follows:

an upstream primer 3: t cell receptor alpha variable region-TRAV _ F1(SEQ ID NOS: 142 to 186, wherein SEQ ID NOS: 183, 177 may also be used alone as TRAV _ F1 for preparing variable region fragments of the alpha genes of C44 and C45, respectively)

An upstream primer 4: t cell receptor alpha variable region-TRAV _ F2(SEQ ID NOS: 42-55, 187 to 214, wherein SEQ ID NOS: 52, 47 may also be used alone as TRAV _ F2 for preparing variable region fragments of the alpha genes of C44 and C45, respectively)

A downstream primer 3: t cell receptor alpha constant region-TRAC 2-GTTGCTCTTGAAGTCCATAGACCTC (SEQ ID NO: 140)

A downstream primer 4: t cell receptor alpha constant region-TRAC 3-CAGGGTCAGGGTTCTGGATA (SEQ ID NO: 141)

Specifically, according to the instructions of the PCR polymerase KOD-Plus-Neo product, the PCR system is 20 μ L in one round, the annealing temperature is 60 ℃, and the reaction is carried out for 30 cycles. Taking 1 mu L of the product of the first round of PCR reaction as a template of the second round of PCR, wherein the second round of PCR system is 30 mu L, the annealing temperature is 60 ℃, and the reaction is carried out for 30 cycles. And then, running agarose gel on the second round PCR product, cutting and recovering the bands with corresponding sizes (Tiangen gel recovery kit), and sequencing, wherein the sequencing primer is a downstream primer 4. Obtaining the TCR alpha gene sequence. Specifically, the alpha chain nucleotide sequences of C44 and C45 are shown as SEQ ID NO: 40. shown at 41.

Second, construction of pHAGE-TCR vector

TCR beta, fp2A and TCR alpha are subjected to overlap-PCR amplification (KOD-Plus-Neo, TOYOBO) by a long primer (containing fp2A sequence) to obtain a TCR beta-fp 2A-TCR alpha fragment which is named as C44 or C45 respectively.

The amplified primers comprise an upstream primer 5(SEQ ID NO: 14(C44), SEQ ID NO: 15(C45)), a downstream primer 5 (shown as SEQ ID NO: 16), an upstream primer 6(SEQ ID NO: 17(C44), SEQ ID NO: 18(C45)), and a downstream primer 6 (shown as SEQ ID NO: 19).

Specifically, primer 5 and primer 6 are used to amplify TCR beta and TCR alpha respectively, PCR system is 50 mu L, annealing temperature is 60 ℃, and reaction is carried out for 30 cycles. And (3) running the PCR product to recover gel (a Tiangen gel recovery kit), taking 1 mu L of the recovered product as a template, and performing overlap PCR by using an upstream primer 5 and a downstream primer 6, wherein the PCR system is 50 mu L, the annealing temperature is 60 ℃, and the reaction is carried out for 30 cycles. The agarose gel was run to obtain a band of about 1800bp, which was recovered by cutting the gel.

The lentiviral vector pHAGE-IRES-RFP was double digested with NotI and NheI. The enzyme cutting system is 40 mu L, wherein the NotI and NheI content is 1.5 mu L respectively, the plasmid is 2-3 mu g, the enzyme cutting is carried out for 6h at 37 ℃, then 1 mu L alkaline phosphatase (NEB) is added into the system, the treatment is carried out for 1h to reduce the plasmid self-ligation, the plasmid after the enzyme cutting is recovered by running gel, and the concentration is measured by using nanodrop and used as a framework for constructing the plasmid.

According to the product instruction of Clone Express II One Step Cloning kit, the TCR is connected with pHAGE-IRES-RFP vector which is linearized after enzyme digestion through overlap (see figure 3), transformed into Stbl3 strain, cultured on LB plate containing aminobenzyl for 12-16h, monoclonal bacteria are picked for sequencing, and sequencing primers are selected from seq-pHAGE-F and seq-pHAGE-R on pHAGE vector and downstream primer 4. The corresponding TCRs were obtained as C44 (the nucleotide sequence is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 30), and C45 (the nucleotide sequence is shown in SEQ ID NO: 33, and the amino acid sequence is shown in SEQ ID NO: 32), respectively.

Example 3: detection of membrane expression and affinity of TCR by pMHC tetramer staining method

1. Construction of endogenous TCR knockout Jurkat T cell lines

Based on the sequence characteristics of the TCR of Jurkat cells, the guide sequences were designed in the constant regions of the alpha and beta chains (TRA _ oligo1-CACCGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 20), TRA _ oligo2-AAACGCCGTGTACCAGCTGAGAGAC (SEQ ID NO: 21), TRB _ oligo1-CACCGGGCTCAAACACAGCGACCTC (SEQ ID NO: 22), TRB _ oligo2-AAACGAGGTCGCTGTGTTTGAGCCC (SEQ ID NO: 23)).

The synthesized guide sequences of the alpha chain and the beta chain are respectively constructed into sgRNA-LentiCRISPR-puro and sgRNA-LentiCRISPR-BSD lentiviral vectors, the vectors and packaging plasmids psPAX2, pMD2.G and PEI transfection reagents are co-transformed into 293T cells according to a certain proportion, cell culture supernatants of 48h and 72h are collected, and the two concentrated viruses simultaneously infect a human Jurkat T cell line. The infection was followed 48h before drug kill with puromycin and blasticidin at appropriate concentrations until all cells in the control group of each of the two drugs died. Surviving cells were cultured in 96-well plates using flow sorting of single cells. For the obtained monoclonal cell line, the expression of the monoclonal cell line is respectively identified by using antibodies of a TCR alpha chain and a TCR beta chain, and the cell line with defects of both chains is the obtained endogenous TCR knockout Jurkat T cell which is named JC 5.

2. Construction of JC5 cell line stably integrating CMV-TCR

The pHAGE-TCR plasmids such as C44 and C45 constructed in example 2 were mixed with the packaging plasmids psPAX2, pMD2.G and PEI as a transfection reagent at a certain ratio, and 293T cells were transfected. Cell culture supernatants were harvested at 48h and 72h and concentrated to infect JC5 cells in the logarithmic growth phase (MOI 0.3). After infection for 3 days, cells are stained by anti-human CD3 and anti-human TCR alpha beta flow antibodies, and cells with the same TCR expression level are sorted and then cultured to obtain the JC5-TCR cell line.

3.TCR Membrane Performance and affinity assays

Take 1X 10⁶JC5-TCR cells with Brilliant Violet 421^TManti-human TCR α β (Biolegend) and the corresponding CMVpp65pMHC Tetramer-APC (Tetramer-APC) were stained prior to flow analysis.

From FIGS. 4 and 5, it can be seen that the prepared specific C44-TCR and C45-TCR against CMV pp65 HLA-A1101 ATVQGQNLK can be correctly expressed and displayed outside the cell membrane, and have a certain affinity with the corresponding tetramer probe. The results show that the TCR prepared by the embodiment of the application has good affinity.

Example 4: construction and in-vitro function detection of human primary TCR-T cells

1. Isolation, culture and lentivirus infection of human primary T cells

To further verify the recognition and killing function of the screened TCR on the CMV pp65 antigen, mononuclear cells (PBMC) were isolated from peripheral blood of volunteers using lymphocyte separation medium Ficoll, T cells were obtained from PBMC by negative selection according to the EasySep Human T cell isolation kit (stem cell technologies) product instructions, and the cells were resuspended to 1X 10 in 1640 complete medium containing 100U/mL IL2⁶cells/mL, cultured in anti-CD 3/CD28 antibody coated dishes for activation. After 48h of activation, the TCR-loaded viral particles (prepared from example 2) were infected with T cells using a lentivirus system by centrifugation at 1500rpm for 2h at 32 ℃ and then removed and cultured in a 37 ℃ cell incubator for 10h, after which the infection was terminated by media exchange and continued at 37 ℃ cell incubator. Three days after infection, TCR positive cells can be sorted by flow cytometry to obtain TCR-T cells (including C44 and C45 as described above).

2. Construction of target cells

Virus particles loaded with pp65-PURO, HLA-A1101-BSD and luciferase-GFP, respectively, were infected with a lentivirus system to Raji cells in log phase growth. Raji cells stably expressing pp65, HLA-A1101 molecule and luciferase-GFP are obtained through drug screening and flow sorting, and are named as Raji-A1101-pp 65-luciferase.

In vitro functional validation of TCR in human Primary T cells

Raji cells (designated Raji) of Raji/HLA1101/pp65 and untransformed pp65 were contacted with 1G4-TCR-T, C44-TCR-T, C45-TCR-T cells in the following ratio of 1: 1, and collecting cells and supernatant after 24 hours of co-culture, and preliminarily detecting the activation condition of C44-TCR-T and C45-TCR-T cells and the death condition of target cells. From the release level of extracellular cytokine IFN γ (see FIG. 6), C44-TCR-T and C45-TCR-T cells were significantly activated after co-incubation with target cells, compared to control group 1G 4-TCR-T. And the amount of luciferase released after lysis from the target cells reflects that C44-TCR-T and C45-TCR-T can significantly kill the target cells (see FIG. 6). Experimental results prove that the C44-TCR-T, C45-TCR-T cell constructed in the embodiment of the invention can be specifically activated by a CMV pp65 antigen peptide presenting cell and can obviously kill a target cell.

Meanwhile, the hypervariable region of CDR3 of the α and β chains of the TCR had the greatest effect on TCR function. It has been experimentally confirmed that the hypervariable region of CDR3, even if highly similar, differs by only one amino acid in function by a very different amount. For example, the amino acids at the first and second positions of CDR3 regions of the α chain and β chain of TCR E141 are mutated sequentially to alanine, and constructed on a lentiviral vector to prepare corresponding TCR-T cells. Co-culturing the cells with target cells at a ratio of 1: 1, and detecting the amount of luciferase and the secreted amounts of cytokines TNF alpha, IL2 and IFN gamma. The results show that the non-mutated E141 can effectively eliminate target cells after encountering HLA matched-Raji, and can specifically produce a large amount of cytokine IL 2. Furthermore, mutations of a single amino acid at the positions a3, a5, a6 and b6, b7, b8 are sufficient to completely inactivate the TCR-T cells (see in particular patent CN 202010373100.4). Accordingly, the mutated TCR, although differing by only one amino acid, is sufficient to completely inactivate the TCR-T cells. It follows that although the target is the same, a difference in only one amino acid will result in two nearly identical TCRs with completely different technical effects, let alone multiple amino acids.

Example 5: animal model construction and CMV TCR-T in vivo function detection

Will be 5X 10⁵Raji-HLA-a 1101-pp 65-luciferase tumor cells were inoculated via tail vein into 5-6 weeks NOD/Scid IL-2R γ null (ncg) female mice, and a lymphoma model was constructed (see fig. 7), which was designated as day 1, and at day 6, the mice were divided into 3 groups, a: PBS injection group (equal volume of PBS injected); b: control NC cell injection group (NC-T cells); c: CMV-TCR-T injection group (C45-TCR-T cell), group B mice tail vein injection 1 x 10⁷NC-T cell, group C mice tail vein injection 1X 10⁷TCR-T cells, group A injected an equal volume of 200. mu.L PBS. Specific reinfusion amounts and reinfusion times of other TCR-T cells are shown in FIG. 7. The growth of tumor cells, proliferation of T cells and survival of mice in the mice were monitored for the next several weeks, and as shown in FIGS. 7-9, CMV-specific C45-TCR-T cells constructed according to the examples of the present invention were able to significantly kill tumor cells in mice as compared with the control group.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 22

caccgggctc aaacacagcg acctc 25

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aaacgaggtc gctgtgtttg agccc 25

<210> 24

<211> 294

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Met Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro

1 5 10 15

Gly Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr

20 25 30

Gln Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro

35 40 45

Arg Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu

50 55 60

Thr Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly

65 70 75 80

Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile

85 90 95

Gln Ile Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg

100 105 110

Gly Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn

115 120 125

Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr

130 135 140

Lys Arg Lys Trp Glu Ala Ala His Ala Ala Glu Gln Gln Arg Ala Tyr

145 150 155 160

Leu Glu Gly Arg Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly

165 170 175

Lys Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His

180 185 190

His Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly

195 200 205

Phe Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp

210 215 220

Gln Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly

225 230 235 240

Thr Phe Gln Lys Trp Val Ala Val Val Val Pro Ser Gly Glu Glu Gln

245 250 255

Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr

260 265 270

Leu Arg Trp Glu Leu Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln

275 280 285

Lys Ile Glu Trp His Glu

290

<210> 25

<211> 885

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgggtagcc atagcatgcg ctacttctat accagcgtga gtcgtccggg ccgtggtgaa 60

cctcgcttta ttgccgtggg ctatgtggat gacacccagt tcgtgcgctt tgatagcgat 120

gccgcaagtc agcgcatgga accgcgtgcc ccgtggattg aacaagaagg cccggaatat 180

tgggaccaag aaacacgcaa cgttaaggcc cagagccaga cagatcgtgt ggatctgggt 240

acactgcgcg gctattacaa tcagagcgag gatggcagcc acaccattca gatcatgtac 300

ggctgcgatg ttggtccgga cggtcgcttt ttacgtggct accgtcaaga tgcctatgat 360

ggcaaggact atatcgcttt aaacgaggat ctgcgcagtt ggaccgcagc cgatatggcc 420

gcacagatca ccaaacgcaa atgggaagca gcacacgccg ccgagcagca acgtgcatat 480

ttagaaggtc gttgcgtgga atggctgcgc cgctatctgg agaatggcaa agaaacttta 540

cagcgtaccg atccgccgaa aacccatatg acccatcacc cgatcagcga tcatgaggcc 600

actttacgtt gttgggcact gggcttttat ccggcagaaa tcactttaac ttggcagcgc 660

gatggtgaag atcagaccca agataccgaa ctggtggaaa cccgccccgc tggtgatggc 720

acctttcaga aatgggttgc agtggttgtg ccgagcggcg aggaacagcg ttacacttgt 780

cacgttcagc atgaaggttt accgaaaccg ctgactttac gttgggaact gggatccggt 840

ttgaacgaca tcttcgaagc tcagaaaatc gaatggcacg aataa 885

<210> 26

<211> 101

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Met Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro

1 5 10 15

Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe

20 25 30

His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile

35 40 45

Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe

50 55 60

Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr

65 70 75 80

Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys

85 90 95

Trp Asp Arg Asp Met

100

<210> 27

<211> 306

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

atggcaatcc agcgtactcc aaagattcag gtttactcac gtcatccagc agagaatgga 60

aagtcaaatt tcctgaattg ctatgtgtct gggtttcatc catccgacat tgaagttgac 120

ttactgaaga atggagagag aattgaaaaa gtggagcatt cagacttgtc tttcagcaag 180

gactggtctt tctatctctt gtactacact gaattcaccc ccactgaaaa agatgagtat 240

gcctgccgtg tgaaccatgt gactttgtca cagcccaaga tagttaagtg ggatcgagac 300

atgtaa 306

<210> 28

<211> 561

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly

1 5 10 15

Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr

20 25 30

Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val

35 40 45

Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp

50 55 60

Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr

65 70 75 80

Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn

85 90 95

Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr

100 105 110

Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val

115 120 125

His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val

130 135 140

Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg

145 150 155 160

Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys

165 170 175

Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp

180 185 190

Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met

195 200 205

Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val

210 215 220

Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu

225 230 235 240

Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met

245 250 255

Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe

260 265 270

Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser

275 280 285

His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu

290 295 300

Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu

305 310 315 320

Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr

325 330 335

Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp

340 345 350

Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr

355 360 365

Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr

370 375 380

Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp

385 390 395 400

Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys

405 410 415

Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Ser Ala Ser Thr Ser

420 425 430

Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ala

435 440 445

Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro

450 455 460

Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala

465 470 475 480

Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu

485 490 495

Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu

500 505 510

Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu

515 520 525

Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln

530 535 540

Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg

545 550 555 560

Gly

<210> 29

<211> 1686

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atggagtcgc gcggtcgccg ttgtcccgaa atgatatccg tactgggtcc catttcgggg 60

cacgtgctga aagccgtgtt tagtcgcggc gatacgccgg tgctgccgca cgagacgcga 120

ctcctgcaga cgggtatcca cgtacgcgtg agccagccct cgctgatcct ggtgtcgcag 180

tacacgcccg actcgacgcc atgccaccgc ggcgacaatc agctgcaggt gcagcacacg 240

tactttacgg gcagcgaggt ggagaacgtg tcggtcaacg tgcacaaccc cacgggccga 300

agcatctgcc ccagccaaga gcccatgtcg atctatgtgt acgcgctgcc gctcaagatg 360

ctgaacatcc ccagcatcaa cgtgcaccac tacccgtcgg cggccgagcg caaacaccga 420

cacctgcccg tagccgacgc tgttattcac gcgtcgggca agcagatgtg gcaggcgcgt 480

ctcacggtct cgggactggc ctggacgcgt cagcagaacc agtggaaaga gcccgacgtc 540

tactacacgt cagcgttcgt gtttcccacc aaggacgtgg cactgcggca cgtggtgtgc 600

gcgcacgagc tggtttgctc catggagaac acgcgcgcaa ccaagatgca ggtgataggt 660

gaccagtacg tcaaggtgta cctggagtcc ttctgcgagg acgtgccctc cggcaagctc 720

tttatgcacg tcacgctggg ctctgacgtg gaagaggacc taacgatgac ccgcaacccg 780

caacccttca tgcgccccca cgagcgcaac ggctttacgg tgttgtgtcc caaaaatatg 840

ataatcaaac cgggcaagat ctcgcacatc atgctggatg tggcttttac ctcacacgag 900

cattttgggc tgctgtgtcc caagagcatc ccgggcctga gcatctcagg taacctgttg 960

atgaacgggc agcaaatctt cctggaggta caagcgatac gcgagaccgt ggaactgcgt 1020

cagtacgatc ccgtggctgc gctcttcttt ttcgatatcg acttgttgct gcagcgcggg 1080

cctcagtaca gcgagcaccc caccttcacc agccagtatc gcatccaggg caagcttgag 1140

taccgacaca cctgggaccg gcacgacgag ggtgccgccc agggcgacga cgacgtctgg 1200

accagcggat cggactccga cgaagaactc gtaaccaccg agcgtaagac gccccgcgtc 1260

accggcggcg gcgccatggc gagcgcctcc acttccgcgg gccgcaaacg caaatcagca 1320

tcctcggcga cggcgtgcac ggcgggcgtt atgacacgcg gccgccttaa ggccgagtcc 1380

accgtcgcgc ccgaagagga caccgacgag gattccgaca acgaaatcca caatccggcc 1440

gtgttcacct ggccgccctg gcaggccggc atcctggccc gcaacctggt gcccatggtg 1500

gctacggttc agggtcagaa tctgaagtac caggagttct tctgggacgc caacgacatc 1560

taccgcatct tcgccgaatt ggaaggcgta tggcagcccg ctgcgcaacc caaacgtcgc 1620

cgccaccggc aagacgcctt gcccgggcca tgcatcgcct cgacgcccaa aaagcaccga 1680

ggttga 1686

<210> 30

<211> 616

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Met Gly Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His

35 40 45

Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro

65 70 75 80

Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Phe Arg Gly Glu Gly Asn Gln Pro Gln His Phe Gly Asp Gly

115 120 125

Thr Arg Leu Ser Ile Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Phe Arg Arg Lys Arg Ser Gly Ser Gly Ala

305 310 315 320

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

325 330 335

Gly Pro Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr

340 345 350

Cys Leu Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu

355 360 365

Met Ser Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp

370 375 380

Thr Ser Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser

385 390 395 400

Arg Gln Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn

405 410 415

Ala Thr Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser

420 425 430

Phe Ser Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr

435 440 445

Phe Cys Ala Tyr Arg Ala Phe Tyr Thr Gly Ala Asn Ser Lys Leu Thr

450 455 460

Phe Gly Lys Gly Ile Thr Leu Ser Val Arg Pro Asp Ile Gln Asn Pro

465 470 475 480

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

485 490 495

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

500 505 510

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg

515 520 525

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

530 535 540

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

545 550 555 560

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

565 570 575

Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val

580 585 590

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

595 600 605

Met Thr Leu Arg Leu Trp Ser Ser

610 615

<210> 31

<211> 1851

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct ttttccgggg cgaggggaat 360

cagccccagc attttggtga tgggactcga ctctccatcc tagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttccggcgga aacggagcgg aagcggagct 960

actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctatggca 1020

tgccctggct tcctgtgggc acttgtgatc tccacctgtc ttgaatttag catggctcag 1080

acagtcactc agtctcaacc agagatgtct gtgcaggagg cagagaccgt gaccctgagc 1140

tgcacatatg acaccagtga gagtgattat tatttattct ggtacaagca gcctcccagc 1200

aggcagatga ttctcgttat tcgccaagaa gcttataagc aacagaatgc aacagagaat 1260

cgtttctctg tgaacttcca gaaagcagcc aaatccttca gtctcaagat ctcagactca 1320

cagctggggg atgccgcgat gtatttctgt gcttataggg ccttttatac tggagccaat 1380

agtaagctga catttggaaa aggaataact ctgagtgtta gaccagatat ccagaaccct 1440

gaccctgccg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc 1500

accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca 1560

gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg 1620

agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 1680

accttcttcc ccagcccaga aagttcctgt gatgtcaagc tggtcgagaa aagctttgaa 1740

acagatacga acctaaactt tcaaaacctg tcagtgattg ggttccgaat cctcctcctg 1800

aaagtggccg ggtttaatct gctcatgacg ctgcggctgt ggtccagctg a 1851

<210> 32

<211> 617

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His

35 40 45

Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe

50 55 60

Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro

65 70 75 80

Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn

85 90 95

Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Leu Ala Gly Tyr Lys Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Leu Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Phe Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr

305 310 315 320

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

325 330 335

Pro Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln

340 345 350

Pro Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys

355 360 365

Gln Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu

370 375 380

Asn Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys

385 390 395 400

Lys Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile

405 410 415

Lys Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser

420 425 430

Ala Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser

435 440 445

Ala Val Tyr Phe Cys Ala Ala Ser Ala Trp Asn Thr Gly Asn Gln Phe

450 455 460

Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val Ile Pro Asn Ile Gln Asn

465 470 475 480

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

485 490 495

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln

500 505 510

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met

515 520 525

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys

530 535 540

Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu

545 550 555 560

Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val

565 570 575

Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser

580 585 590

Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

595 600 605

Leu Met Thr Leu Arg Leu Trp Ser Ser

610 615

<210> 33

<211> 1854

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60

gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120

agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180

ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240

ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300

gagctggggg actcggccct ttatctttgc gccagcagct tggcgggata caagcaagag 360

acccagtact tcgggccagg cacgcggctc ctggtgctcg aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttcttc cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggctttac ctcggtgtcc taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcctgctagg gaaggccacc ctgtatgctg tgctggtcag cgcccttgtg 900

ttgatggcca tggtcaagag aaaggatttc cggcggaaac ggagcggaag cggagctact 960

aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tatggccatg 1020

ctcctggggg catcagtgct gattctgtgg cttcagccag actgggtaaa cagtcaacag 1080

aagaatgatg accagcaagt taagcaaaat tcaccatccc tgagcgtcca ggaaggaaga 1140

atttctattc tgaactgtga ctatactaac agcatgtttg attatttcct atggtacaaa 1200

aaataccctg ctgaaggtcc tacattcctg atatctataa gttccattaa ggataaaaat 1260

gaagatggaa gattcactgt tttcttaaac aaaagtgcca agcacctctc tctgcacatt 1320

gtgccctccc agcctggaga ctctgcagtg tacttctgtg cagcaagcgc gtggaacacc 1380

ggtaaccagt tctattttgg gacagggaca agtttgacgg tcattccaaa tatccagaac 1440

cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 1500

ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 1560

acagacaaaa ctgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 1620

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 1680

gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 1740

gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 1800

ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag ctga 1854

<210> 34

<211> 311

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Met Gly Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His

35 40 45

Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro

65 70 75 80

Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Phe Arg Gly Glu Gly Asn Gln Pro Gln His Phe Gly Asp Gly

115 120 125

Thr Arg Leu Ser Ile Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Phe

305 310

<210> 35

<211> 310

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His

35 40 45

Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe

50 55 60

Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro

65 70 75 80

Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn

85 90 95

Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Leu Ala Gly Tyr Lys Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Leu Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Phe

305 310

<210> 36

<211> 278

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu

1 5 10 15

Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys

100 105 110

Ala Tyr Arg Ala Phe Tyr Thr Gly Ala Asn Ser Lys Leu Thr Phe Gly

115 120 125

Lys Gly Ile Thr Leu Ser Val Arg Pro Asp Ile Gln Asn Pro Asp Pro

130 135 140

Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys

145 150 155 160

Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp

165 170 175

Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met

180 185 190

Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe

195 200 205

Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe

210 215 220

Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser

225 230 235 240

Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly

245 250 255

Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr

260 265 270

Leu Arg Leu Trp Ser Ser

275

<210> 37

<211> 280

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro

1 5 10 15

Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln

20 25 30

Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn

35 40 45

Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys

50 55 60

Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys

65 70 75 80

Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala

85 90 95

Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala

100 105 110

Val Tyr Phe Cys Ala Ala Ser Ala Trp Asn Thr Gly Asn Gln Phe Tyr

115 120 125

Phe Gly Thr Gly Thr Ser Leu Thr Val Ile Pro Asn Ile Gln Asn Pro

130 135 140

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

145 150 155 160

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

165 170 175

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg

180 185 190

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

195 200 205

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

210 215 220

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

225 230 235 240

Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val

245 250 255

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

260 265 270

Met Thr Leu Arg Leu Trp Ser Ser

275 280

<210> 38

<211> 933

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct ttttccgggg cgaggggaat 360

cagccccagc attttggtga tgggactcga ctctccatcc tagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttc 933

<210> 39

<211> 930

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60

gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120

agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180

ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240

ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300

gagctggggg actcggccct ttatctttgc gccagcagct tggcgggata caagcaagag 360

acccagtact tcgggccagg cacgcggctc ctggtgctcg aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttcttc cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggctttac ctcggtgtcc taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcctgctagg gaaggccacc ctgtatgctg tgctggtcag cgcccttgtg 900

ttgatggcca tggtcaagag aaaggatttc 930

<210> 40

<211> 837

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga atttagcatg 60

gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga gaccgtgacc 120

ctgagctgca catatgacac cagtgagagt gattattatt tattctggta caagcagcct 180

cccagcaggc agatgattct cgttattcgc caagaagctt ataagcaaca gaatgcaaca 240

gagaatcgtt tctctgtgaa cttccagaaa gcagccaaat ccttcagtct caagatctca 300

gactcacagc tgggggatgc cgcgatgtat ttctgtgctt atagggcctt ttatactgga 360

gccaatagta agctgacatt tggaaaagga ataactctga gtgttagacc agatatccag 420

aaccctgacc ctgccgtgta ccagctgaga gactctaaat ccagtgacaa gtctgtctgc 480

ctattcaccg attttgattc tcaaacaaat gtgtcacaaa gtaaggattc tgatgtgtat 540

atcacagaca aaactgtgct agacatgagg tctatggact tcaagagcaa cagtgctgtg 600

gcctggagca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag cattattcca 660

gaagacacct tcttccccag cccagaaagt tcctgtgatg tcaagctggt cgagaaaagc 720

tttgaaacag atacgaacct aaactttcaa aacctgtcag tgattgggtt ccgaatcctc 780

ctcctgaaag tggccgggtt taatctgctc atgacgctgc ggctgtggtc cagctga 837

<210> 41

<211> 843

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60

agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120

gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180

tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240

gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300

ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgcg 360

tggaacaccg gtaaccagtt ctattttggg acagggacaa gtttgacggt cattccaaat 420

atccagaacc ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct 480

gtctgcctat tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat 540

gtgtatatca cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt 600

gctgtggcct ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt 660

attccagaag acaccttctt ccccagccca gaaagttcct gtgatgtcaa gctggtcgag 720

aaaagctttg aaacagatac gaacctaaac tttcaaaacc tgtcagtgat tgggttccga 780

atcctcctcc tgaaagtggc cgggtttaat ctgctcatga cgctgcggct gtggtccagc 840

tga 843

<210> 42

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

gaagaaagga cgaataagtg ccac 24

<210> 43

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

acatcacagc cacccagact ac 22

<210> 44

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

cacagaagac agaaagtcca gcac 24

<210> 45

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gcaatcgctg aagacagaaa gtc 23

<210> 46

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gaaaggacag ttctctccac atcac 25

<210> 47

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

aaaagtgcca agcacctctc tc 22

<210> 48

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gcaaagctcc ctgtacctta cg 22

<210> 49

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

aaagataact gccaagttgg atgag 25

<210> 50

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

cttcctgaat atctcagcat ccatac 26

<210> 51

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

gcatcctgaa catcacagcc ac 22

<210> 52

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

gaatcgtttc tctgtgaact tccag 25

<210> 53

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

tctcagcacc ctccacatca c 21

<210> 54

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

caggtatcag actcagccgt gtac 24

<210> 55

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

gccacaataa acatacagga aaagc 25

<210> 56

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

atggatacct ggctcgtatg c 21

<210> 57

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

atgggctgca ggctcctc 18

<210> 58

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

atgggctgca ggctgctc 18

<210> 59

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

atgggctcca ggctgctct 19

<210> 60

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

atgggccccg ggct 14

<210> 61

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

atgggccctg ggctcct 17

<210> 62

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

atgggaccca ggctcctct 19

<210> 63

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

atgagcatcg ggctcctgtg 20

<210> 64

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

atgagcctcg ggctcctg 18

<210> 65

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

atgagaatca ggctcctgtg c 21

<210> 66

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

atgagcatcg gcctcctgtg 20

<210> 67

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

atgagcatca gcctcctgtg 20

<210> 68

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

atgggcacaa ggctcctctg 20

<210> 69

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

atgggcacca ggctcctc 18

<210> 70

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

atgggcacca gtctcctatg c 21

<210> 71

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

atgggtacca gtctcctatg ctg 23

<210> 72

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

atgggcacca gcctcctc 18

<210> 73

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

atgggcttca ggctcctctg 20

<210> 74

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

atgggcacga ggctcttctt c 21

<210> 75

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

atgggcacca ggctcttctt c 21

<210> 76

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

atgggcacaa ggttgttctt c 21

<210> 77

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

atgagcacca ggcttctctg c 21

<210> 78

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

atgggtacca ggctcctctg 20

<210> 79

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

atggactcct ggaccttctg ct 22

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

atggactcct ggaccctctg 20

<210> 81

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

atggccacca ggctcctc 18

<210> 82

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

atgcttagtc ctgacctgcc tg 22

<210> 83

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

atggtttcca ggcttctcag tt 22

<210> 84

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

atgggtcctg ggcttctcc 19

<210> 85

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

atgagcccaa tattcacctg ca 22

<210> 86

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

atggatatct ggctcctctg ct 22

<210> 87

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

atggacacca gagtactctg ctg 23

<210> 88

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

atgagcaacc aggtgctctg 20

<210> 89

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

atgctgctgc ttctgctgc 19

<210> 90

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

atggcctccc tgctcttctt c 21

<210> 91

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

atgactatca ggctcctctg ctac 24

<210> 92

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

atgggccccc agctcc 16

<210> 93

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

atgggaatca ggctcctctg tc 22

<210> 94

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

atgctgagtc ttctgctcct tctc 24

<210> 95

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

atgctctgct ctctccttgc 20

<210> 96

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

tcactctgaa gatccggtcc ac 22

<210> 97

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

cttcacatca attccctgga gc 22

<210> 98

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

cacctgaatg ccccaacag 19

<210> 99

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

agtcgcttct cacctgaatg c 21

<210> 100

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

gagatgaatg tgagcacctt ggag 24

<210> 101

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

gagatgaatg tgagtgcctt ggag 24

<210> 102

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

gctctgagct gaatgtgaac gc 22

<210> 103

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

gaagtcccca atggctacaa tg 22

<210> 104

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

aggtccctga tggctacaat g 21

<210> 105

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

tggcgtctgc tgtaccctct 20

<210> 106

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

acagaggatt tcccgctcag 20

<210> 107

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

cccctcaagc tggagtcagc 20

<210> 108

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

acacagagga tttcccactc agg 23

<210> 109

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

ttctctgcac agaggtctga gg 22

<210> 110

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

gctgcccagt gatcgcttct 20

<210> 111

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

actctgaaga tccagcgcac ag 22

<210> 112

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

cgtctccact ctgaagatcc agc 23

<210> 113

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

ggcctgaggg atccatctcc 20

<210> 114

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

ggctgctcag tgatcggttc 20

<210> 115

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

gaacgattct ccgcacaaca gt 22

<210> 116

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

acaaaggaga agtctcagat ggct 24

<210> 117

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

ccctcactct ggagtcagct ac 22

<210> 118

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

cctcactctg gagtccgcta c 21

<210> 119

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

gcagagaggc tcaaaggagt agac 24

<210> 120

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

atccagccct cagaacccag 20

<210> 121

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

atcattctga actgaacatg agctc 25

<210> 122

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

tggagggacg tattctactc tgaag 25

<210> 123

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

gacacccctg ataacttcca atcc 24

<210> 124

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

tgcctcccaa attcaccctg 20

<210> 125

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

catcccgcag agccgag 17

<210> 126

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

gcaggtagtg cgaggagatt c 21

<210> 127

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

ctgtgacatc ggcccaaaag 20

<210> 128

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

acagtgacca gtgcccatcc 20

<210> 129

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

caatgcccca agaacgcac 19

<210> 130

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

tagagtctgc catccccaac c 21

<210> 131

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

gacggagcat tttcccctga c 21

<210> 132

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

gccagcacca accagacatc 20

<210> 133

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

ctgatcctgg agtcgcccag 20

<210> 134

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

atcagccgcc caaacctaac 20

<210> 135

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

cggcagttca tcctgagttc taag 24

<210> 136

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

tcaggcagta tctggagtca ttg 23

<210> 137

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

gcacctcctt cccattcacc 20

<210> 138

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

gcttctgatg gctcaaacac ag 22

<210> 139

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

cgaccagctt gacatcacag 20

<210> 140

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

gttgctcttg aagtccatag acctc 25

<210> 141

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

cagggtcagg gttctggata 20

<210> 142

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

atgtggggag ctttccttct ct 22

<210> 143

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

atgtggggag ttttccttct ttatg 25

<210> 144

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

atggctttgc agagcactct g 21

<210> 145

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

atggcctctg cacccatctc 20

<210> 146

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

atgaggcaag tggcgagagt 20

<210> 147

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

atgaagacat ttgctggatt ttcg 24

<210> 148

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

atggagtcat tcctgggagg tg 22

<210> 149

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

atggagaaga tgcggagacc tg 22

<210> 150

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

atgctcctgt tgctcatacc ag 22

<210> 151

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

atgctcctgc tgctcgtcc 19

<210> 152

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

atgctcctgg agcttatccc a 21

<210> 153

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

atgctcttag tggtcattct gctg 24

<210> 154

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

atgaattctt ctccaggacc agc 23

<210> 155

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

atgaactatt ctccaggctt agtatctc 28

<210> 156

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

atgaaaaagc atctgacgac cttc 24

<210> 157

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

atgatatcct tgagagtttt actggtg 27

<210> 158

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

atgaaatcct tgagagtttt actagtgatc 30

<210> 159

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

atgatgaaat ccttgagagt tttactgg 28

<210> 160

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

atgacatcca ttcgagctgt atttat 26

<210> 161

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

atggcaggca ttcgagctt 19

<210> 162

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

atgtcacttt ctagcctgct gaag 24

<210> 163

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

atgaagccca ccctcatctc ag 22

<210> 164

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

atggaaactc tcctgggagt gtc 23

<210> 165

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

atgctgtctg cttcctgctc ag 22

<210> 166

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

atgctgactg ccagcctgt 19

<210> 167

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

atggagaaaa tgttggagtg tgc 23

<210> 168

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

atggagaccc tcttgggcct 20

<210> 169

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

atgaagagga tattgggagc tctg 24

<210> 170

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

atggacaaga tcttaggagc atcat 25

<210> 171

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

atgctgttct ccagcctgct 20

<210> 172

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

atggagaaga atcctttggc ag 22

<210> 173

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

atgctactca tcacatcaat gttgg 25

<210> 174

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

atgaggctgg tggcaagagt aac 23

<210> 175

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

atgaagttgg tgacaagcat tactg 25

<210> 176

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

atggtcctga aattctccgt gtc 23

<210> 177

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

atggccatgc tcctggg 17

<210> 178

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

atggagactc tcctgaaagt gct 23

<210> 179

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

atggagactg ttctgcaagt actcc 25

<210> 180

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

atgctccttg aacatttatt aataatct 28

<210> 181

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 181

atgatgaagt gtccacaggc tttac 25

<210> 182

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 182

atgacacgag ttagcttgct gtg 23

<210> 183

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 183

atggcatgcc ctggcttc 18

<210> 184

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 184

atgaagaagc tactagcaat gattctg 27

<210> 185

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 185

atgaactcct ctctggactt tctaattc 28

<210> 186

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 186

atggtgaaga tccggcaatt 20

<210> 187

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 187

tccttagtcg ctctgatagt tatggt 26

<210> 188

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 188

tttcttcatt ccttagtcgg tctaaag 27

<210> 189

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 189

ctcttcatcg ctgctcatcc tc 22

<210> 190

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 190

gccaaacctc cttccacctg 20

<210> 191

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

cctgccgaca gaaagtccag 20

<210> 192

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 192

cgcattgcag acacccagac 20

<210> 193

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 193

ttgataccac ccttaaacag agtttg 26

<210> 194

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 194

attactgaag aatggaagca gcttg 25

<210> 195

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 195

tcctttaatc tgaggaaacc ctctg 25

<210> 196

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 196

gtgaaacctc cttccacctg ac 22

<210> 197

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 197

aggctttgag gctgaattta agag 24

<210> 198

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 198

ggctgaattt aacaagagtc aaacttc 27

<210> 199

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 199

gagcgaaacc tccttctacc tg 22

<210> 200

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 200

aaaccacttc tttccacttg gag 23

<210> 201

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 201

caaagcaaag ctctctgcac atc 23

<210> 202

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 202

cagtgattca gccacctacc tc 22

<210> 203

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 203

cgaattgctg ttacattgaa caagac 26

<210> 204

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

ttgcagctac tcaacctgga gac 23

<210> 205

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

atccgccaac cttgtcatct c 21

<210> 206

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 206

aaggcgagac atctttccac c 21

<210> 207

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

agattaagag tcacgcttga cacttc 26

<210> 208

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 208

gccagtccta tcaagagtga cagttc 26

<210> 209

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 209

caacttcacc atcacagcct cac 23

<210> 210

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 210

cacatcacag cccctaaacc tg 22

<210> 211

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 211

tggaagactt aatgcctcgc tg 22

<210> 212

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 212

tgtcgctacg gaacgctaca g 21

<210> 213

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 213

cttcaataaa agtgccaagc agttc 25

<210> 214

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 214

aaaaagtggt cgctattctg tcaac 25

<210> 215

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 215

Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

1 5 10 15

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 216

<211> 81

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 216

cggcggaaac ggagcggaag cggagctact aacttcagcc tgctgaagca ggctggagac 60

gtggaggaga accctggacc t 81