WO2011038689A1 - Novel human cytokine vstm1-v2 and use thereof - Google Patents

Abstract

The present invention provides a novel human cytokine VSTM1-v2, its encoding gene or its immunity fragments, use of said protein, gene, or its immunity fragments in promoting Th17 differentiation and the killing function of CD8+T lymphocytes, and use of said protein, gene or its immunity fragments in preparing pharmaceutical composition for prevention and/or treatment of immune related diseases. The present invention also provides the VSTM1-v2 antagonists such as monoclonal antibodies or polyclonal antibodies, vector, host cell or composition comprising VSTM1-v2, and reagent for testing VSTM1-v2 or its immunity fragments, and use thereof.

Description

 Human new cytokine VSTM1-V2 and its application

 The present invention relates to a novel human cytokine and its use, in particular to a VSTM1-V2 gene or protein of VSTM1 or an immunological fragment thereof, the VSTM1-V2 gene or protein or their The use of immunological fragments in the killing function of Thl7 and CD8+ T lymphocytes and in the preparation of pharmaceutical compositions for the treatment of immune-related diseases, and also on antagonists of VSTM1-V2, including VSTM1-V2 Vector, host cell or composition, and reagents for detecting VSTM1-V2 or an immunological fragment thereof, and their use. Background technique

 The immune system plays a vital role in the body. It can prevent the invasion of pathogenic microorganisms externally, and it can clear the cells of aging, disease and death in time, and maintain the stability of the internal environment. The immune system survives the above functions in response to an immune response. Cytokine secreted by the cell can regulate cell growth and differentiation, regulate immune function and physiological response, and participate in pathological reactions. Cytokines are generally small molecule secreted proteins that, through binding to cytokine receptors, transmit information between immune cells and play important regulatory roles in both innate and adaptive immune responses. Up to now, more than 200 cytokines have been discovered, which can be divided into six categories according to their functions: 1. Interleukin (IL); 2. Colony-Stimulating Factor (CSF); Interferon (IFN); 4, Tumor-Necrosis Factor (TNF); 5, Chemokine (CK); 6, Growth Factor (GF). There are a wide variety of cytokines and a wide range of functions.

 Th cells are the hub of adaptive immune responses, and cytokines are the primary means by which they work. Th cells can be divided into

Thl, Th2 and Thl7, which produce characteristic cytokines are IFN, IL-4 and -17, respectively. Thl's main role is to protect against infection by intracellular pathogenic microorganisms, to participate in the development and development of autoimmune diseases such as rheumatoid arthritis and diabetes; Th2 is involved in anti-parasitic infections and allergic reactions; Thl7 is resistant to extracellular bacteria and fungi It plays an important role in infection and participates in the occurrence and development of autoimmune diseases such as inflammatory bowel disease (Jinfang Zhu, William E. Paul, CD4 T cells: fates, functions, and faults, Blood, 2008, 112 (5): 1557-1568). As a new subpopulation of CD4+ effector T cells, ΤΜ7 cells mainly secrete cytokines such as IL-17A, IL-17F, IL-22, IL-26 and tumor necrosis factor. These inflammatory factors mediate inflammatory responses (protection against infection by extracellular pathogens), autoimmune diseases, tumors and transplant rejection. Related studies have found that rheumatoid arthritis (RA), multiple sclerosis (MS), asthma, lupus, and IL-17 expression in transplant rejection are both increased. Studies have found that a large number of IL-17+ cells are present in the intestinal mucosa of patients with autoimmune inflammatory bowel disease such as ulcerative colitis (UC) and Crohn's disease (CD). IL-17 |3⁄4 plays an important role in the induction and maintenance of mucosal inflammatory response in inflammatory bowel disease, and also promotes the secretion of various inflammatory cytokines, such as IL26, TNF-β, CC family chemokines. Recent studies have also found blood in patients with vasculitis, multiple sclerosis, nephrotic syndrome, psoriasis, etc. In Qinghe tissues, the expression level of IL-17 is closely related to the course of disease and disease (Lauren A. Zenewicz, Andrey Antov, Richard A. Flavell, CD4 T-cell differentiation and inflammatory bowel disease, Trends in Molecular Medicine, 2009, 15 (5) ): 199-207; Jennifer Louten, Katia Boniface, Rene de Waal Malefyt, Development and function of TH17 cells in health and disease, J Allergy Clin Immumol, 2009, 123 (5): 1004-1011). Recent studies have found that many cytokines are involved in the regulation of differentiation of TM7 cells. It is generally believed that IL-6 and TGF-β provide the initiating factor for the differentiation of primary T cells into TM7 cells. IL-23 plays a very important role in maintaining differentiation into TM7 cells; inflammatory cells such as TNF-o IL-Ιβ Factor can promote the differentiation of Th17 cells; TM7 cells can secrete IL-21, which has self-feedback effect; while IL-25, IL-27 and IL-35 show more inhibition of TM7 cell differentiation, but its effect may be More complicated. In-depth study of the differentiation, physiological and pathological functions and regulatory mechanisms of TM7 cells has important theoretical significance and potential application value for the study of autoimmune diseases.

 CD8+ T cells can recognize MHC-I/antigen peptide complexes and kill target cells. They are also known as killer T cell CTLs. Their main function is to eliminate host cells infected by viruses and other intracellular parasitic microorganisms. It was demonstrated that tumor-specific CTL can kill the corresponding tumor cells. Upon activation of CD8+ T cells, perforin can be released and the cytokines TNF-α and IFN-γ secreted, which in turn kill target cells.

 At present, recombinant cytokines or recombinant soluble receptors produced by genetic engineering technology and therapeutic antibodies are used to treat tumors,

»The barriers, infections, etc. have received good results and become a new generation of drugs. Recombinant cytokines have many advantages as drugs, such as: cytokines are the body's own components, can regulate the body's physiological processes and improve immune function, can play a very low dose, so the effect is significant, side effects are small, is a A new biologic has become an indispensable treatment for some difficult conditions (Antonella Viola, Andrew D. Luster, Chemokines and Their Receptors: Drug Targets in Immunity and Inflammation, Annual Review of Pharmacology and Toxicology, 2008, 48: 171 -197). Currently approved cytokine drugs include interferon alpha, beta, gamma, Epo, GM-CSF, G-CSF, IL-2 and the like. In addition, according to incomplete statistics, at least 26 international genomic drugs have entered clinical research, including new recombinant cytokines and recombinant soluble receptors. At the same time, cytokine or cell surface receptor detection is also an important indicator for judging the body's immune function and immune cell differentiation. It has important laboratory research value and has many practical value in clinical practice, including diagnosis of many diseases, disease and disease. , efficacy judgment and cytokine treatment monitoring. Summary of the invention

 It is an object of the present invention to provide a splicing body of VSTM1, particularly a protein of VSTM1-V2 or a derivative thereof or an immunological fragment thereof.

 Another object of the present invention is to provide a polynucleotide sequence encoding a protein of VSTM1-V2 or a protein derived therefrom or an immunological fragment thereof.

 Another object of the invention is to have a carrier comprising VSTM1-V2.

Another object of the invention is to provide a host cell comprising VSTM1-V2. Another object of the invention is to have an antagonist of VSTM1-V2.

 Another object of the invention is the use of a gene or protein of VSTM1-V2 or an immunological fragment thereof for the killing function of the Z7 differentiation and/or CD8+ T lymphocytes.

 Another object of the present invention is the use of a gene or protein of VSTM1-V2 or an immunological fragment thereof, or an antagonist thereof, for the preparation of a composition for preventing and/or treating diseases of the immune system.

 Another object of the present invention is to provide a composition for preventing and/or treating an immune related disease.

 Another object of the invention is to use a reagent for detecting genes or proteins of VSTM1-V2 or immunological fragments thereof.

 Another object of the invention is the use of reagents for the co-detection of genes and proteins of VSTM1-V2 or immunological fragments thereof. The present invention comprises the following proteins (a) or (b) or immunological fragments thereof:

 (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an immunological fragment thereof; or

 (b) a protein derived from (a) or an immunological fragment thereof which has been substituted, deleted or added with one amino acid in (a) a defined amino acid sequence and which has the same function as (a);

 The immunological fragment is preferably a polypeptide consisting of the amino acid sequence shown in positions 17 to 32 or 62 to 81 of SEQ ID NO: 4, or an amino acid sequence shown in positions 17 to 205 of SEQ ID NO: 4. protein.

 The present invention also provides a polynucleotide sequence encoding the protein of (a) or (b) above or an immunological fragment thereof; wherein the polynucleoside is preferably a polynucleotide sequence of SEQ ID NO: 3.

 The invention also provides a genetically engineered vector comprising the polynucleotide sequence; the vector is preferably a plasmid. The invention also provides a host cell obtained by transformation, transfection or transduction of said genetically engineered vector.

 The invention also provides an antagonist against said protein or an immunological fragment thereof or said polynucleotide sequence; preferably said antagonist is an antibody, antisense RNA or small interference R A .

 The invention also provides the use of the protein or an immunological fragment thereof or the polynucleotide sequence for promoting Thl7 differentiation and/or killing function of CD8+ T lymphocytes. For example, the protein or an immunological fragment thereof or the polynucleotide sequence is used in the preparation of a preparation for promoting the killing function of Thl7 differentiation and/or CD8+ T lymphocytes.

 The present invention also provides the use of the protein or an immunological fragment thereof, the polynucleotide sequence, or the antagonist for preventing and/or treating an immune-related disease, for example, in preparation for prevention and And the use of the pharmaceutical composition for treating an immune-related disease; wherein the protein is preferably a protein consisting of the amino acid sequence shown in positions 17 to 205 of SEQ ID NO: 4, and the polynucleotide sequence is preferably SEQ. ID NO: The polynucleotide sequence shown by 3; the antagonist is preferably a monoclonal antibody or a polyclonal antibody; and the immune-related disease is preferably an infectious disease, an autoimmune disease or a tumor. The polynucleotide sequence may be contained in a vector; the vector is preferably a plasmid.

The present invention also provides a pharmaceutical composition for preventing and/or treating an immune-related disease, the composition comprising the protein or an immunological fragment thereof, or the polynucleotide sequence, or the Vector, host cell, or antagonist An agent; and one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers.

 The invention also provides an agent for detecting the protein or an immunological fragment thereof or the polynucleotide sequence.

 The present invention also provides assays for detecting a polynucleotide sequence of said protein or an immunological fragment thereof, for example, for use in the preparation of a composition for aided diagnosis and prognosis of immune-related diseases. Preferably, the agent is an antibody, antisense R A or small interfering RNA. The immune-related disease may specifically be an infectious disease, an autoimmune disease, a sword, or the like.

 The present inventors have found that there are at least five splicing bodies of human gene K3⁄4M, namely VSTMl-vl, VSTM1-V2, VSTMl-v3, VSTMl-v4, VSTMl-v5. Among them, VSTMl-v2 encodes a classical secretory protein containing N-glycosylation modification sites, which promotes the killing function of TM7 differentiation and CD8+ T lymphocytes; Description VSTM1-V2 is a new potential cytokine. Therefore, the gene or protein of VSTM1-V2 or its immunological fragment, antibody, siRNA has potential clinical application value in infectious diseases, autoimmune diseases, adjuvant diagnosis, prevention and/or treatment of tumors. DRAWINGS

 Figure 1 shows the results of analysis of the characteristics of VSTM1-V2's affinity to ice (1), antigenicity (2), and surface exposure (3) using the DNAstar software package. The rectangular box in the figure indicates the site of the polypeptide design in Example 5 of the present invention.

2A, 2B and 2C show expression profiling RT-PCR analysis of VSTM1-v2 of the present invention in normal tissues. Figure 2A shows a library of 16 normal tissues of humans, each lane being: 1- brain; 2-heart; 3-kidney; 4-liver; 5-lung; 6-pancreas; 7-placenta; 8-skeletal muscle; - colon; 10-white blood cells; 11-ovary; 12-prostate; 13-small intestine; 14-spleen; 15-testis; 16-thymus. Figure 2B shows a library of seven normal immune system tissues of humans, each lane being: 1-white blood cells; 2-bone marrow; 3-lymph nodes; 4-spleen; 5-thymus; 6-tonils; 7-fetal liver; Figure 2C shows the specific expression profiles of VSTM1-V2, each lane being: 1-brain; 2-heart; 3-kidney; 4-liver; 5-lung; 6-pancreas; 7-placenta; 8-skeletal muscle; - colon; 10-white blood cells; 11-ovary; 12-prostate; 13-small intestine; 14-spleen; 15-testicular; 16-thymus; 17-bone; 18-lymph; 19- tonsils; 21-pcDB-VSTMl-v2 _; 22- pcDB-VSTMl-vl

 Figure 3A shows the purification of GST-VSTM1-v2 recombinant protein by SDS-PAGE and mass spectrometry in Example 4 and ? Enzyme digestion results. Among them, lanes 1 and 2 are GST-VSTM1-V2 recombinant protein samples before and after enzyme digestion, respectively. The right side of the picture shows the results of the mass spectrometry of the corresponding strip. Fig. 3B shows the results of purification of the Trx-His-S-VSTM1-v2 recombinant protein by SDS-PAGE in Example 4. Among them, lane 1 is the supernatant of bacterial lysis, and lanes 2 and 3 are components eluted from the nickel column during protein purification.

 Figure 4 shows the results of Western blot analysis of the specificity of the VSTM1 antibody prepared as a prokaryotic protein in Example 5. Among them, lane 1 was a negative control group transfected with empty vector, and lane 2 was an experimental group transfected with pcDB-VSTM1-v2.

Figure 5 shows the super-span of VSTMl-v2 protein by Western blot; the effect of BFA on the secretion of VSTMl-v2. Figure 6 shows the results of SDS-PAGE and Western blot to identify the purified protein of VSTM1-v2 secreted protein in eukaryotic cells. Among them, lanes 1, 2, 3, 4, and 5 are SDS-PAGE results, lane 6 is Western blot, lanes 1, 2, and 6 are purified VSTM1-V2 proteins, and lanes 3, 4, and 5 are BSA protein standards. Figure 7 shows the glycosylation modification of VSTMl-v2 secreted protein purified by eukaryotic cells by Western blot. Lane 1 is a negative control to which no enzyme is added, Lane 2 is added to the N-glycosidase F group, and Lane 3 is added to the 0-glycosidase group.

 Figure 8 shows the effect of VSTM1-V2 secreted protein on IL-4C images of CD4+ T cells by flow cytometry A), IFN-γ (; picture B), IL-17AC picture C) cytokine expression. Reference numerals 1 to 8 in the figure represent the isotype control group, PBS, VSTMl-v2-l, VSTMl-v2-10 VSTMl-v2-100 AVSTMl-v2-100 rVSTMl-v2 ArVSTMl-v2 group, respectively.

Figure 9 shows the results of examining the effect of VSTM1-V2 secreted protein on differentiation of TM7 cells in Example 9. Among them, picture A, TM7 intracellular cytokine staining, showing the percentage of IL-17A positive cells and mean fluorescence intensity; Figure B, ELISA analysis of secreted IL-17A concentration; Picture C, IL-17A and RORC mRNA levels after Thl7 cell differentiation Change; Figure D shows the results of cell proliferation assayed by [ ³ H]-TdR incorporation assay. Donor 1 and Donor 2 represent two different individuals. Reference numerals 1 to 3 in pictures B, C, and D represent PBS, VSTMl-v2-VSTMl-v2-10, respectively.

 Figure 10 shows the effect of detecting VSTM1-V2 on the killing function of CD8+ T cells. Among them, picture A shows the results of FACS detection of intracellular cytokine IFN-γ; picture B is an optical micrograph of CD8+ T cells and K562 cells; Figure C shows the use of flow cytometry Annexin V-FITC/PI double staining Apoptosis results of K562 cells. Reference numerals 1 and 2 in the figure represent the PBS and VSTM1-V2-10 groups, respectively.

 Figure 11 shows the results of the identification of VSTM1-V2 transgenic mice. Among them, picture A, PCR was identified at the genome level; Picture B, ELISA detected VSTMl-v2 in serum, wherein the left panel shows the standard curve of VSTMl-v2 by indirect ELISA; Figure C, Western blot. WT is a wild type mouse, n=4, Transgenic is a VSTMl-v2 transgenic mouse, n=8.

 Figure 12 shows the results of HE staining of tissues of wild type and VSTM1-V2 transgenic mice. Among them, pictures A, B, C, D, I, J, K, L and £, F, G, H, M, N, 0, P show the heart, liver and spleen of wild-type and VSTMl-v2 transgenic mice, respectively. , lungs, kidneys, brain, testes and skeletal muscle tissue. WT is a wild type mouse and Transgenic is a VSTM1 -v2 transgenic mouse.

 Figure 13 shows peripheral blood granulocytes and mononucleosis in VSTM1-V2 transgenic mice. Among them, picture A shows the blood routine analysis of each mouse; picture B shows the percentage of white blood cells and their absolute values of lymphocytes, monocytes and granulocytes, respectively. Among them, LY is lymphocyte, MO is monocyte, GR is granulocyte, WT is wild type mouse, n=4, TG is VSTM1-V2 transgenic mouse, n=8.

 Figure 14 shows elevated levels of IFN-γ and IL-17A in the serum of VSTMl-v2 transgenic mice. WT is a wild type mouse, n=4, Transgenic is a VSTMl-v2 transgenic mouse, n=8.

 Figure 15 shows that VSTM1-V2 promotes the expression of IFN-γ and IL-17A in spleen cells of transgenic mice. Picture A, spleen cell surface marker immunofluorescence analysis; Picture B, spleen cell intracellular cytokine staining, left and right images show the percentage of positive cells and average fluorescence intensity; Figure C, ELISA analysis of spleen cells secreted cytokines. WT was wild type mouse, n=4, TG was VSTMl-v2 transgenic mouse, n=8.

Figure 16 shows that VSTM1-V2 transgenic mice accelerate the onset of the EAE model. Which picture A, wild type mice and Clinical score of VSTM1-V2 transgenic mouse EAE model; Panel B, percentage of mice after immunization with MOG; Panel C, highest clinical score for each mouse during observation; Figure D, HE staining of spinal cord sections of EAE-infected mice, Arrows indicate mature lymphocyte infiltration; Figure E, FACS detects Th cell subsets in spleen cells of EAE mice, and the left and right images are the percentage of positive cells and the mean fluorescence intensity, respectively. WT was wild type mice, n=9, TG was VSTM1-V2 transgenic mice, n=6.

 Figure 17 shows the expression level of VSTM1-V2 in the serum of patients with rheumatoid arthritis. detailed description

 The present invention found that the human novel gene VSTM1 has at least five splicing bodies, namely VSTM1-vl, VSTM1-v2, VSTM1-v3 VSTM1-v4, VSTM1-v5 (see Example 1). Among them, VSTMl-v2 encodes a classical secretory protein, i.e., the VSTM1-V2 protein of the present invention.

 According to an aspect of the present invention, the present invention provides the protein represented by the following (a) or (b) or an immunological fragment thereof:

 (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an immunological fragment thereof; or

 (b) A protein derived from (a) or an immunological fragment thereof which has been substituted, deleted or added with one or several amino acids in (a) a defined amino acid sequence and which has the same function as (a).

 The amino acid sequence as set forth in SEQ ID NO: 4 is the protein sequence of VSTM1-V2 of the present invention, which has a total of 205 amino acids, a molecular weight of 22.5 kD, and an isoelectric point of 4.84. The protein has two N-glycosylation sites and has a signal migratory column (SEQ ID No: 4, positions 1 to 16). Since the TMHMM assay has no transmembrane region, VSTM1-v2 may be a new secreted protein.

 The immunological fragment of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 may be any immunogenic fragment of the VSTM1-V2 protein, for example, positions 17 to 32 or 62 to 81 of SEQ ID NO: A polypeptide consisting of the amino acid sequence shown, or a protein consisting of the amino acid sequence shown in positions 17 to 205 of SEQ ID NO: 4. According to the present invention, the VSTM1-V2 protein or an immunological fragment thereof can be substituted, deleted or added with one or several amino acids to obtain the same function as the VSTM1-V2 protein or an immunological fragment thereof. Sequence derived from a sex fragment. Techniques for substituting, deleting or adding one or several amino acids to obtain derivatives having the same function are known to those skilled in the art, for example, may be between non-polar amino acids or between polar amino acids (especially not Substitution of charged polar amino acids or between polar amino acids with the same charge (positive or negative charge). For example, one skilled in the art can utilize the classification of common amino acids to perform substitutions between non-polar amino acids or between polar amino acids, such as the exchange between Asp and Glu.

 The present invention also provides a polynucleotide sequence encoding the protein of (a) or (b) above or an immunological fragment thereof.

The VSTM1-V2 gene is a polynucleotide sequence encoding SEQ ID NO: 4 of the present invention, which may be a coding sequence of the amino acid represented by SEQ ID NO: 4, and may include non-coding in addition to the coding sequence of the above amino acid sequence. A sequence, such as an intron, a non-coding sequence at the 5' or 3' end of the coding sequence, and the like. The polynucleotide sequence may be DNA or RA, wherein the DNA includes cDNA, genomic DNA, and synthetic DNA, and may be single-stranded or double-stranded, and may be a coding strand or a non-coding strand. A preferred gene is the polynucleotide sequence of SEQ ID NO: 3, which is 640 nucleotides in length, and Contains sequences encoding the VSTM1-V2 protein (eg, coding sequence (CDS: nucleoside 12 to 629) and 5' non-coding region (nucleotides 1 to 11) and 3' non-coding region (nucleotide No. 630~640)). Alternatively, an isolated nucleotide sequence which comprises only the coding sequence of the VSTM1 protein is more preferred.

 It is known to those of ordinary skill in the art that the nucleotide sequence of VSTM1-V2 of the present invention may be identical to the coding sequence set forth in SEQ ID NO: 3, or may be completely identical to the above-described nucleus due to the degeneracy of the genetic code. The coding sequence of the nucleotide. For example, depending on the frequency of codons used by each particular prokaryotic or eukaryotic host, the corresponding codon can be selected to increase the efficiency of expression of the polynucleotide in the corresponding host. Codons can also be converted in order to obtain polynucleotides that have better performance than native nucleotide sequences, such as longer half-lives.

 The immunological fragment of the VSTM1-V2 gene or protein of the present invention includes an immunological fragment of the VSTM1-V2 protein of the present invention or an immunological fragment of the VSTM1-V2 gene. The immunological fragment of the VSTM1-V2 gene of the present invention may be a nucleotide sequence encoding an immunological fragment of the VSTM1-V2 protein, for example: an amino group represented by positions 17 to 205 of SEQ ID NO: 4, a multinuclear column The nucleotide sequence, preferably the polynucleotide sequence shown in positions 60 to 629 of SEQ ID NO: 3.

 The protein or immunological fragment thereof, and the polynucleotide sequence provided by the present invention are isolated proteins or immunological fragments thereof, and polynucleotide sequences. By "isolated" is meant that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, a polynucleotide or a protein or polypeptide amino acid sequence in a natural state in a living cell is not isolated and purified, but the same polynucleotide or protein or polypeptide is separated if it is separated from other substances coexisting in the natural state. Purified. Such polynucleotides may be part of a vector, and it is also possible that such polynucleotides or proteins or multiple months are part of a composition that is not a component of their natural environment, these multinuclei The nucleotide or protein polypeptide is still isolated.

 The polynucleotide sequences of the present invention can be obtained by methods available in the art. These techniques include, but are not limited to: (1) isolation of DNA sequences by hybridization techniques; (2) artificial chemical synthesis of DNA sequences; (3) large-scale acquisition of desired polynucleotides by construction of cDNA libraries; (4) PCR amplification technology. For example, the polynucleotide sequence of VSTM1-V2 of the present invention or an immunological fragment thereof can be obtained by using cDNA, mRNA or genomic DNA as a template according to standard PCR amplification techniques, and selecting appropriate oligonucleotide primers for amplification. Thus, the guanosine can be cloned into a vector and then obtained by replication in the vector. It can also be obtained by standard DNA synthesis techniques, for example, on a DNA synthesizer using a solid phase phosphite triester method well known in the art. The gene of the present invention, or a variety of DNA fragments, can be assayed by conventional methods such as dideoxy chain termination (Sanger et al. PNAS, 1977, 74: 5463-5467); commercial sequencing can also be used. Box and so on. In order to obtain a full-length cDNA sequence, sequencing needs to be repeated. It is sometimes necessary to determine the cDNA sequences of multiple clones in order to splicing into full-length cDNA sequences.

The VSTM1-V2 protein of the present invention or an immunological fragment thereof may be a recombinant protein or polypeptide, a natural protein or polypeptide, a synthetic protein or polypeptide, a semisynthetic protein or polypeptide, a recombinant protein or polypeptide. The protein of the invention or an immunological fragment thereof may be a naturally purified product, or a chemically synthesized product, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). . The protein of the present invention or an immunological fragment thereof may be glycosylated or A non-glycosylated; may or may not include an initial methionine residue.

 The VSTM1-V2 protein of the present invention or an immunological fragment thereof can be obtained by a conventional method, for example, according to Steward, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, 111. The method described in (1984)) is synthesized by solid phase chemistry using an Applied Biosystem synthesizer or a PioneerTM synthesizer, or can be produced in a cell-free translation system using rnRNA derived from the DNA construct of the present invention. Protein or polypeptide. A protein or polypeptide product can also be produced by recombinant DNA sequences in a host cell according to conventional bioengineering methods (Science, 1984; 224: 1431), for example, comprising the steps of: (1) using a polynucleotide of the invention (or Variant), or transforming, transfecting or transducing a suitable host cell with an expression vector containing the polynucleotide; (2) cultivating the host cell obtained in step (1) in a suitable medium; (3) culturing from the culture The desired protein or polypeptide is isolated or purified in the cell or cell.

The polynucleotides and proteins of the invention or immunological fragments thereof are preferably provided in isolated form, more preferably purified to homogeneity. According to another aspect of the invention, the invention provides a vector comprising a polynucleotide sequence of the invention. The polynucleotide sequence of the present invention can be inserted into a recombinant expression vector. The genetic engineering vector may be a general vector, an expression vector or the like. The common vector is mainly used for the establishment of various genomic libraries and cDNA libraries, which usually contain two or more marker genes, one of which is used to select transformants (transfonnant) and the other gene is used for check vectors. Is there any foreign DNA insertion? Expression vectors are mainly used to study gene expression or to mass produce some useful transcription products or proteins, and some can also be used for the establishment of cDNA libraries. In addition to the characteristics of a conventional vector, such vectors should contain an appropriate promoter, ribosome binding site, terminator, and the like. To facilitate localization of the purine product in the cell, an appropriate leader sequence can be added upstream of the polypeptide coding sequence. If necessary, in order to increase the transcription efficiency of the DNA encoding VSTM1-V2 of the present invention in higher eukaryotic cells, an enhancer sequence can be inserted into the vector. Selection of suitable vectors and promoters is well known to those skilled in the art. Methods for constructing vectors comprising the polynucleotides of the invention and suitable transcriptional and translational regulatory components are well known to those skilled in the art. Specifically, the term "recombinant expression vector" refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses, such as adenoviruses, retroviruses, or other vectors well known in the art. The vector suitable for use in the present invention may be a prokaryotic expression vector or an eukaryotic expression vector. Commercially available expression vectors suitable for use in prokaryotic cells generally carry a selectable marker and a cell origin of replication, with bacterial promoters such as lad, T7 (Rosenberg, et al. Gene, 1987, 56: 125), PL and trP, and Other genetic components of the cloning vector pBR322 (ATCC 37017) are known. Such commercially available vectors, including pGEM (Promega) and pKK223-3 (Phannacia), can be selected from the appropriate vectors derived from pBR322 depending on the appropriate promoter selected and the structural gene sequence to be expressed. A GST prokaryotic expression system can also be used in the present invention. Vectors suitable for eukaryotic cells carry eukaryotic promoters such as CMV, SV40, etc. Such vectors include pM-hlLi (Ma Dalong, Di Chunhui, Pang Jian et al. (1991) High Technology News 11: 26-29), pQE-9 (Qiagen), pD10, pNHI 8A (Stratagene), pKK233-3 pDR540, pRrr5 (Pharmacia), and pcDNATM3.1/myc-hisB (-) (Invitrogen), pCI, pWLNEO pSG (Stratagene), pSVL ( Pharnlacia), pcDNA3. Ι 5-His-TOPO (Invitrogen, hereinafter abbreviated as pcDT). The present invention prefers pcDT, which can be directly ligated with a PCR product to construct a eukaryotic expression vector, which greatly improves the efficiency of large-scale production. In a specific embodiment of the invention, the PCR product of the VSTM1-V2 polynucleoside is cloned into pGEM-T Easy (Promega), pcDNA3.1/mycHis (-) B pGEX4T-l and pET-32a-c (+) vectors. Any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control component. The expression vector containing the polynucleotide sequence of the present invention and the transduction control signal can be constructed using a square wire well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant DNA techniques, and the like (Sambrook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).

 The present invention also relates to a host cell which is genetically engineered by the above vector or the polynucleotide of the present invention and which is suitable for expressing the protein of the present invention or an immunological fragment thereof. The vectors and polynucleotides of the invention can be used to transform a suitable host cell such that it is capable of expressing a protein of a human secretory cytokine. Such hosts include, but are not limited to, prokaryotic hosts, such as Escherichia coli, Bacillus, Streptomyces, etc.; eukaryotic hosts, such as: Saccharomyces, Aspergillus, insect cells, various fly s2 and grass 繊S©; plant cells Animal cells, such as CHO, COS (monkey kidney fibroblast cell line, Gluzman (Cell 23: 175, 1981)) or Bowes melanoma cells, 293 T, HeLa cells, and other cell lines capable of expressing a compatible vector.

Those skilled in the art will know how to select appropriate vectors, promoters, enhancers and host cells and methods for introducing a construct comprising a polynucleotide of the present invention into such host cells, including but not limited to: calcium chloride mediated Transformation, calcium phosphate transfection, DEAE-15 dextran mediated transfection, electroporation, microinjection, particle bombardment or gene gun method (Sambrook, ! (1989), Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press), after the transformed host strain or cell is cultured in appropriate culture conditions and culture medium, and grown to the appropriate cell density, the selected start is induced by an appropriate method (eg, transformation or chemical induction). Son, and culture the cells for a while. It is within the knowledge of one of ordinary skill in the art to select the appropriate culture conditions and media for the different host strains or cells and the nature of the protein or polypeptide of interest. When the host cell is a prokaryotic cell such as E. coli, competent cells capable of absorbing DNA can be collected after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. An alternative is MgCl ₂ treatment, which can also be treated by electroporation. When the host is a eukaryotic cell, the following transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. The obtained transformant can be cultured by a conventional method to express the protein or polypeptide encoded by the polynucleotide of the present invention. The appropriate conventional medium is selected according to the host cell of choice, and cultured under conditions suitable for the growth of the host cell. Host cells used in the examples of the present invention are, for example, Escherichia coli BL21 and 293T cells (ATCC CRL-11268) and the like.

The invention also provides a method of producing the protein or an immunological fragment thereof: culturing a host cell comprising a polynucleotide encoding the invention or a fragment thereof, under conditions suitable for expression; obtaining from the cell culture A protein or polypeptide encoded by a polynucleotide. The recombinant protein or polypeptide in the above method may be coated intracellularly, extracellularly or expressed on the cell membrane or secreted extracellularly. If desired, the recombinant protein or polypeptide can be isolated and purified by various separation methods using its physical, chemical, and other properties. Specifically, after the host cell is transformed and the transformed host cell is grown to a suitable cell density, the promoter is induced by an appropriate method (e.g., variation or chemical trait induction), and then the culture is continued. After the completion of the culture, the cells can be collected by centrifugation and used by any known method, which is well known to those skilled in the art, such as freeze-thaw method, sonication, osmotic bacteria, The cells are disrupted by lysozyme dissolution or mechanical disruption. The protein or polypeptide of the present invention or a fragment or fusion protein or polypeptide thereof can be recovered and purified from host cell culture by various known methods, including iron sulfate or ethanol precipitation, acid extraction, ultracentrifugation, super Filtration, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, gel over affinity chromatography, high performance liquid chromatography and various other liquid chromatography techniques or combinations of these methods .

 The invention also relates to nucleic acid fragments homologous to any portion of a polynucleotide of the invention. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. This nucleic acid fragment is usually a DNA sequence chemically synthesized based on the nucleotide sequence information of the present invention. The above nucleic acid fragments can be used in PCR amplification techniques (e.g., as primers) to identify and/or isolate polynucleotides encoding human secretory cytokines; they can also be used as probes for hybridization. It can also be used for RNA interference technology. A part or all of the polynucleotide of the present invention can also be immobilized as a probe on a microarray or a DNA chip for analyzing differential expression and gene diagnosis of genes in tissues. The label of the probe may be a radioisotope, a fluorescein or an enzyme such as an alkaline phosphatase or the like. Whether or not these fragments encode a protein or polypeptide or a protein or polypeptide encoded has the function of the protein or polypeptide of the present invention is not particularly important for detecting, hybridizing and/or inhibiting expression.

The invention also provides antagonists of the genes or proteins directed against said VSTM1-V2. Antagonists (eg, proteins, nucleic acids, aqueous compounds) can bind to VSTM1-V2 of the invention and inhibit or block the biological activity of VSTM1-V2. Preferably, the antagonist is an antibody, antisense RA or small interfering RNA. The antibody includes a monoclonal antibody or a polyclonal antibody, preferably a neutralizing antibody. The antibody preferably has the sequence shown by amino acid residues 17-205 of SEQ ID NO: 4, the amino acid residues 17 to 32 of SEQ ID NO: 4, and the amino acid residue 62 of SEQ ID NO: 4 A sequence-specific binding polyclonal antibody or monoclonal antibody shown at 81. The "specific binding" refers to a property in which a polyclonal antibody or a monoclonal antibody specifically recognizes a target antigen and binds to a different antigenic epitope or antigenic determinant of the target antigen. The antibodies described in the present invention include those which bind and Antibodies which inhibit the VSTM1 gene product of the invention also include those which do not affect the function of the VSTM1 polypeptide of the invention. The above antibodies include not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab fragments and Antibodies raised by Fab expression libraries; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as having murine antibody binding specificity but still retaining An antibody derived from a human antibody portion. The antibody of the polypeptide protein of the present invention or an immunological fragment thereof can be produced by preparing a square wire using an antibody known in the art. Examples are: Monoclonal antibodies can be produced by hybridoma technology (KoMer and lstein. Nature, 1975 , 256: 495-497). Production of polyclonal antibodies can be immunized with the polypeptide protein of the present invention or an immunological fragment thereof , such as rabbits, mice, rats, etc. In one embodiment of the invention (see Example 5), the antigenic VSTM1 N-terminus (SEQ ID NO: 4 amino acid sequences 17 to 32 and 62) ~81) and VSTM1 prokaryotic protein (amino acid sequence 17-205 of SEQ ID NO: 4) as an example, polyclonal antibody was prepared, antibody titer was detected by ELISA, and antibody specificity was confirmed by Western blot, which confirmed that the titer was high and specific. A good antibody, which can be further used for expression profiling and functional studies of VSTM 1. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Human constant regions and non-human sources can be used. The chimeric antibodies that bind to the variable regions can be produced using existing techniques (Morrison et al. PNAS, 1985, 81: 6851). Single-chain antibodies can also be produced using existing techniques (US Pat No.). 4946778). The various antibodies of the present invention can be obtained by conventional immunological techniques using the proteins of the present invention or immunological fragments, derivatives, analogs or cells expressing the same as the antigen, and these fragments or functional regions can be recombinantly used. Prepared or synthesized using a peptide synthesizer.

 According to one aspect of the present invention, the present invention provides the use of a gene or protein of VSTM1-V2 or an immunological fragment thereof for promoting differentiation of TM7, and a gene or protein of VSTM1-V2 or an immunological fragment thereof Application of the killing function of the ship CD8+T lymphocytes. For example, the use of the VSTM1-V2 gene or protein or immunological fragment thereof for the preparation of a ship Thl7 differentiation and/or a killing function of CD8+ T lymphocytes. Thl7 plays an important role in the prevention of extracellular bacterial and fungal infections, and participates in the occurrence and development of autoimmune diseases such as inflammatory bowel disease. In-depth study of the differentiation, physiological and pathological functions and regulatory mechanisms of TM7 cells, and research on autoimmune Disease has important theoretical significance and potential application value. CD8+ T lymphocytes act as CTLs after activation, killing target cells and playing an important role in anti-tumor and anti-viral immunity. Since the VSTM1-V2 gene or protein of the present invention or an immunological fragment thereof can promote TM7 differentiation and can kill the CD8+ T lymphocyte, and antagonize Qia to inhibit or block the biological activity of VSTM1-V2, The gene or protein of VSTM1-V2 of the present invention or an immunological fragment thereof or an antagonist thereof can be used for the prevention and/or treatment of an immune-related disease (such as an infectious disease, an autoimmune disease, a tumor, etc.), specifically In other words, the genes or proteins of VSTM1-V2 or their immunological fragments can be widely used for the prevention and/or treatment of bacterial fungal and viral infectious diseases, as well as for the prevention and/or treatment of tumors. Neutralizing antibodies, antisense RA or small interfering RNA can be used for the prevention and/or treatment of autoimmune diseases.

 According to another aspect of the present invention, the gene or protein of the VSTM1-V2 of the present invention or an immunological fragment thereof, or an antagonist thereof, is prepared in a pharmaceutical composition for preventing and/or treating an immune-related disease. application. Wherein, the protein is preferably a protein of the amino acid thief shown in positions 17 to 205 of SEQ ID NO: 4, and the polynucleotide sequence is preferably the polynucleotide sequence shown by SEQ ID NO: 3; The antagonist is preferably a monoclonal antibody or a polyclonal antibody; and the immune-related diseases are, for example, infectious diseases, autoimmune diseases, tumors and the like. The genes and proteins of VSTM1-V2 of the present invention may be directly contained in a pharmaceutical composition for treating an immune-related disease in the form of genes and proteins, and may be treated with a transient expression product thereof, or may be contained in an expression vector. The form is contained in a pharmaceutical composition for the treatment of immune related diseases, and the treatment is carried out with transient and stable expression products.

The present invention also provides a pharmaceutical composition for preventing and/or treating an immune-related disease, the composition comprising the protein or an immunological fragment thereof, the polynucleotide sequence, or the vector, or the Said host cell, or said antagonist _; and one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers. Pharmaceutically acceptable excipients or pharmaceutically acceptable carriers refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or other formulation excipients. The pharmaceutical composition is suitable for local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal administration. When treated in the above or other manner, a therapeutically effective amount of the invention

VSTM1-V2 may be in the form of a pure form, a pharmaceutically acceptable salt of the gene or protein of VSTM1-V2 of the invention or an immunological fragment thereof, or alternatively in combination with a pharmaceutically acceptable excipient. The specific therapeutically effective dose for a specific patient for fti depends on a number of factors, including the condition being treated and its severity; the activity of the particular compound employed; the particular composition employed; the age, weight, sex of the patient Another | J, diet and general health; dosing time; route of administration; specific excretion rate; duration of treatment or other drugs taken at the same time. The VSTM1-V2 protein of the present invention or an immunological fragment thereof can also be used by expressing the protein or an immunological fragment thereof in vivo. For example, a patient's cells can be genetically engineered by using a gene encoding a protein of the present invention or an immunological fragment thereof in vitro, and then the engineered cells are supplied to the patient, thereby allowing the engineered cell to express the protein or an immunological fragment thereof in vivo, thereby To achieve the purpose of treatment.

 The present invention also provides a method for detecting whether a change in the gene or protein of the VSTM1-V2 of the present invention in a sample from a test subject is in vitro, comprising: detecting the polynucleotide or protein in the sample to be tested Or the expression level of the polypeptide; comparing the expression level of the polynucleotide or protein or polypeptide in the sample to be tested with the expression level of the polynucleotide or protein or polypeptide of the normal sample; determining the polynucleotide or protein in the sample to be tested Or whether the expression level of the polypeptide changes. The normal sample can be obtained from a cell of a normal human that is not known to be diseased, and the cell should be identical to the tissue source of the sample cell to be tested; the expression level of the polynucleotide of the normal sample can be obtained from the cell of the normal human. The expression level of a statistically significant polynucleotide. The method for detecting the level of a polynucleotide in a sample to be tested may be any of the above detection methods, preferably detecting the expression level of the polynucleotide at a nucleic acid level by RT-PCR; or detecting by using a specific monoclonal or polyclonal antibody; The expression level of the polynucleotide at the protein level, such as immunohistochemistry. The test sample can be obtained from cells from a subject, such as cells from blood, urine, saliva, gastric juice, biopsy, and autopsy material.

 The present invention also provides a test for detecting the gene or protein of VSTM1-V2 of the present invention or an immunological fragment thereof. For example, an agent for detecting expression of the polynucleotide at a nucleic acid level; or an agent for detecting expression of the polynucleotide at a protein level. The present invention also provides the use of an agent for detecting the expression of a gene or protein of VSTM1-V2 or an immunological fragment thereof for the preparation of a composition for aiding diagnosis and prognosis of an immune-related disease. The reagent may be a protein, a nucleic acid, a water compound or the like, and is preferably an antibody, an antisense RNA or a small interfering R A (siRNA). The genes or proteins of VSTMl-v2 of the present invention or immunological fragments thereof can be used as diagnostic indicators. Therefore, the pathological state caused by insufficient or excessive expression of VSTM1-V2 in the present invention can be detected by detecting the expression level of the gene or protein of VSTM1-V2 of the present invention or an immunological fragment thereof, and the specific detection method can be Restriction fragment length polymorphism analysis (RFLP), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), or the like, or a combination thereof. Similarly, the same object can be attained by radioimmunoassay, competitive binding assay, Western blot analysis or enzyme-linked immunosorbent assay (ELISA) using the antibody of the VSTM1-V2 protein of the present invention.

In a specific embodiment of the present invention, the present invention utilizes _P cDB-VSTMl-v2-myc-his eukaryotic cell transfected supernatant, purified recombinant human VSTM1-V2 protein for functional study, and RT-PCR experiment shows VSTMl-v2 Expressed only in the immune system and immune cells, suggesting that VSTM1-V2 plays a major role in the immune system. The invention further finds that VSTMl-v2 can significantly promote the expression of IL-17A in CD4+ T cells by FACS, ELISA, realtime PCR and the like, and VSTMl-v2 can promote the differentiation of Th17 cells in vitro. The [ ³ H]-TdR incorporation assay detected the proliferation of VSTM1-V2 for £3⁄4 Thl7 cells. Moreover, in a specific embodiment of the present invention, it was also found that VSTM1-V2 can significantly promote the expression of CD8+ T cell IFN-γ, and i CD8+ T cell killing effect. VSTM1-V2 may act as a cytokine to mediate differentiation and regulation of CD4+ T lymphocytes The function of CD8+ T lymphocytes plays an important role in the immune system.

 The VSTM1-V2 of the present invention is a secreted protein which can be produced by various cells such as immune system cells and white blood cells, and plays an important regulatory role in an immune system. Therefore, VSTM1-V2 possesses the structural and functional characteristics of cytokines and may play an important role as a cytokine. VSTM1-V2 protein, antibody, siR A have potential clinical application value in infectious diseases, autoimmune diseases, tumor-assisted diagnosis, prevention and/or treatment. Example

 The invention is further illustrated by the following specific examples. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods for specific cattle are not indicated in the following examples, and are generally carried out according to the conditions described in conventional conditions such as Sambrook et al., The Guide to Molecular Cloning (third edition) (Cold Spring Harbor Laboratory (CSHL) Press, 2001). , or in accordance with the conditions recommended by the manufacturer. Example 1. Cloning and bioinformatics analysis of cDNA of VSTM1 gene

 The granulocyte cDNA library was first prepared by the following method: i TRIzol (invitrogen) extracts granulocyte total R A (operating according to the instructions), and reverse transcribed to synthesize a single-strand cDNA library (operating according to the instructions) using Reverse transcriptTM kit CInvitrogen. The above methods for collecting peripheral blood mononuclear cells and granulocytes are described in the literature (Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by receiving centrifugation and sedimentation at Lg. Scandinavian Journal of Clinical and Laboratory Investigation. Supplement (Oslo) 97, 77-89).

The cDNA sequences as well as the alternative splices were analyzed by alignment with NCBI's Human_est database. Chromosomal localization was obtained by NCBI's tBLASTn program while obtaining chromosomal localization from the genomic database ihttp:, www.ncbi.nlm.nih.gov/geiionK. Protein Functional Domain Analysis Using the NCBI CDD Database

/Stmcture/cdd/cdd. shtnil). Other protein-related analyses were performed using the ExPASy database ihttp: 〃 us.expasv.org ), including: TMHMM (http://www.cbs.dto.dk/services/TMHMM'') for transmembrane region analysis; Signal P ( Http :〃www. cbs. dtu. dk /services SignaSP ) for signal makeup analysis; Psort Π (http://psort.nibb.ac.jp) for subcellular localization prediction; GNF SvmAtlas (http://svmatlas .grforg/Sho AtlasA for expression profiling; PrositeOittp:, www.expasv.om/prosite/) for domain analysis. DNAstar software was used to analyze protein antigenicity, hydrophilicity, surface exposure and the like. Through bioinformatics analysis, it was found that UniGene: Hs.444431, its corresponding gene is VSTM1, GenelD: 284415, located at 19ql3.42, which is a new human gene with unknown function. The sequence is corrected by BLASTn method using Human_est database, and then according to This sequence designed a nested specific primer for the full-length reading frame of the VSTM1 gene: the outer forward primer 5'-GCAAGAGTGGGGCAGAG-3' (SEQ ID No: 11); the outer reverse primer 5'-ACGAAGAGCAAGGAAACAC-3, (SEQ ID No: 12); medial forward primer 5'-GAAGGGA CGCTATGACCGC-3' (SEQ ID No: 13); medial reverse primer 5'-CTGTCTTCTTGCTACACTTTC-3, (SEQ ID No: 14). Using the above primers, respectively, a human normal spleen tissue cDNA library (Clontech: Cat. No. 636743), The human normal fetal liver tissue cDNA library (Clontech: Cat. No. 636748) and the human normal granulocyte cDNA library (previously prepared) were subjected to the first PCR amplification reaction as a template, and the reaction conditions were as follows:

The reaction volume is 50μ1, which contains: human normal spleen/fetal liver tissue granulocyte cDNA template: 5μ1 (5ng); Primer: outer forward primer, reverse primer final concentration 0.2μΜ _; dNTP: final concentration 200μΜ; Taq DNA polymerization Enzyme: 2.5 U; lOxTaq DNA polymerase buffer: 5 μl; make up to 50 μl volume with double distilled water.

 Reaction temperature, time: 94 ° C, denaturation for 5 minutes; then denaturation at 94 ° C for 30 seconds, annealing at 52 ° C for 30 seconds, extension at 72 ° C for 1 minute, amplification for 30 cycles; finally extending at 72 ° C for 10 minutes .

 The second PCR amplification reaction was carried out by diluting 50 times with double distilled water as a template. The reaction conditions were as follows: The reaction volume was 50 μl, which contained: 50 μM diluted by a product: 5 μl (5 ng); The medial forward arches were 0.2 μΜ each at the final concentration of the reverse primer; dNTP: 200 μΜ at the final concentration; Taq DNA polymerase: 2.5 U; lOxTaq DNA polymerase buffer: 5 μl; 50 μl volume supplemented with double distilled water.

 Reaction temperature, time: 94 ° C, denaturation for 5 minutes; then denaturation at 94 ° C for 30 seconds, annealing at 61 ° C for 30 seconds, extension at 72 ° C for 1 minute, amplification for 30 cycles; finally extending at 72 ° C for 10 minutes .

 The amplified product was a 3' base A 3' overhanging sticky end fragment, purified using the QIAquick Glue Recovery Kit (Qiagen, 28706) according to the manufacturer's instructions, followed by a 3' base T linear pGEM-T EASY vector (Promega, A1360) was ligated at 16 °C for 8 hours, and the ligated product was transformed into Escherichia coli DH5 (x (commercially available from companies such as TakaRa), and the transformants were grown on LB plate medium containing ampicillin, and clones were selected. Plasmids were extracted and sequenced using an Abl PRISM 3700 DNA Analyzer (Perkin-Elmer/Applied Biosystem).

 In this embodiment, human normal spleen tissue, fetal liver tissue and granulocyte cDNA library are used as templates to amplify VSTM1, and five splicing forms of VSTM1 gene are obtained. The five splicing bodies of the present invention are named VSTMl-vl. VSTM1-v2, VSTM1-v3, VSTM1-v4, VSTM1-v5, the protein sequence and nucleic acid sequence thereof are shown in SEQ ID Nos: 1 to 10. The pGEM-T EASY vectors containing the cDNA of the five splicing bodies of VSTM1 were named pGEM-T-VSTM1-vl, pGEM-T-VSTMl-v2 pGEM-T-VSTMl-v3 pGEM-T-VSTMl-v4 pGEM- T-VSTMl-v5.

The VSTMl-v2 tissue source is spleen and granulocytes. VSTM1-V2 encodes a 205 amino acid protein with a molecular weight of 22.5 kD and an isoelectric point of 4.84. The most characteristic of this protein is that it has a secretion signal J3⁄4 ^ column (SEQ ID No: 4, positions 1 to 16), and Signal P analysis has a distinct signal peptide, which is N-terminal 16 amino acids and has two N-glycosylation sites. TMHMM analysis has no transmembrane region, and VSTM1-V2 may be a new secreted protein. The results of predicting the antigenicity, hydrophilicity, surface exposure and the like of VSTM1-V2 using the DNAstar software package (DNASTAR Inc., Madison, WI, USA) are shown in Fig. 1. Example 2. Expression profile analysis of VSTMl-v2 in tissues and cells

 To analyze the mRNA expression level of VSTM1-V2 in normal tissues, the purchased clontech human normal tissue cDNA library was used in this example, and the nested PCR amplification of the bovine VSTM1 was carried out in Example 1. The 5' primer (5 '-TGAAGGTCGGAGTCAACGGATTTGGT-3 ' SEQ ID No : 15 ) and the 3 ' primer (5'-CATGTG GGCCATGAGGTCCACCAC-3' SEQ ID No: 16) were used to amplify GAPDH as an internal reference, and the amplified bovine was 94. C (5 minutes) → 94. C (40 seconds), 58. C (40 seconds), 72. C (40 seconds), amplification 20 cycles → 72. C (7 minutes). The PCR amplification products were subjected to agarose l electrophoresis, EB staining, and GENE Snap gel imaging system.

 The results of the experiment are shown in Fig. 2A and Fig. 2B. Fig. 2A shows a library of 16 normal tissues of humans. Each lane is divided into IJ: 1, brain; 2, heart; 3, kidney; 4, liver; Lung; 6, pancreas; 7, placenta; 8, skeletal muscle; 9, colon; 10, white blood cells; 11, ovary; 12, prostate; 13, small intestine; 14, spleen; 15, testis; Figure 2B shows a library of seven normal immune system tissues of humans, each lane being: 1, white blood cells; 2, bone marrow; 3, lymph nodes; 4, spleen; 5, thymus; 6, tonsil; 7, fetal liver; Control.

 Since the molecular weight difference between VSTM1-V2 and VSTMl-vl is small, the two are difficult to distinguish in agarose electrophoresis. In order to further clarify the expression profile of VSTM1-V2, the present invention designed a primer for specific amplification of VSTM1-V2, and analyzed the expression spectrum thereof. The results are shown in Fig. 2C, and the VSTMl-vl and VSTMl-v2 plasmids are respectively systematic. Negative control and positive control, the results were in line with expectations, indicating that the designed primers can selectively amplify VSTM1-V2.

 The tissue expression profiling of this example showed that VSTM1-V2 was mainly expressed in immune organs and immune cells such as spleen, thymus, lymph nodes, bone marrow, white blood cells, etc., suggesting that VSTM1-V2 may be an important cytokine of the human body, in the immune system. Play an important role. In addition, VSTM1-V2 also has a certain abundance in the liver, placenta and ovaries.

 Example 3. Construction of eukaryotic expression plasmid pcDB-VSTMl-v2-myc-his

In order to examine the function of VSTM1-V2, a eukaryotic expression plasmid containing VSTM1-V2 cDNA was constructed in this example: pcDNA3. lB-VSTMl-v2-myc-his (pcDB-VSTMl-v2-myc-his). The upstream primer (5'-CGAGCGGCCGCATGACCGCAGAATTCCTCTC-3, SEQ ID No: 17) with a Not I (TaKaRa) cleavage site and the downstream bow of the Kpn I (TaKaRa) cleavage site (5'-CTTGGTACCGACACTTTCAGTGCC GCATATT -3' SEQ ID No: 18) PCR amplification of _P GEM-T-VSTM1-v2 vector (see preparation in Example 1) (reaction temperature, time: 94 V, denaturation for 5 minutes; then denaturation at 94 ° C for 30 seconds , annealing at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute, amplification for 35 cycles; finally at 72 ° C for 10 minutes), obtaining the full-length ORF fragment of VSTM1-V2 cDNA, then using Not I and Kpn I cleave the PCR product and digest the eukaryotic expression vector pcDNA3.1/mycHis (-) B with Not I and Kpn I (Invitrogen, V85520, hereinafter abbreviated as pcDB, pcDB is designed for recombinant protein in mammals A highly expressed vector in a cell line containing the human cytomegalovirus CMV promoter for high expression in mammalian cell lines. The VSTM1-V2 cDNA gene fragment after cleavage is ligated to the vector at 16 °C. In hours, E. coli DH5a was transformed, and the transformant was grown on LB plate medium containing ampicillin. Long colonies, plasmids were extracted, the PCR identified positive clones by sequencing (with Above), select the correct insert sequence of the VSTM1-V2 cDNA gene plasmid, named pcDB-VSTMl-v2-myc-his (with c-myc and his tags). Example 4 Construction of prokaryotic expression plasmid of VSTM1-V2 and purification of prokaryotic protein

 1. Construction of prokaryotic expression plasmid of VSTM1-V2

 In order to prepare a VSTM1 antibody and perform a functional study of the secreted protein VSTM1-V2, a construct was constructed in this example.

Prokaryotic expression vectors for VSTMl-v2 cDNA: pGEX4T-l-VSTMl-v2 and pET-32a-c(+)-VSTMl-v2. The VSTM1-V2 fragment inserted therein is the ORF region after removal of the signal peptide. First, the upstream primer (5'-CGCGGATCCTACGAAGATGAGAAAAAGAATG-3, SEQ ID No: 19) with a BamH I (TaKaRa) cleavage site and the downstream marker of the Sma I (TaKaRa) cleavage site (3'-CGCCGTGACTTTCACATCGGGCCCCCT - 5' SEQ ID No: 20) PCR amplification of _P GEM-T-VSTM1-v2 vector (see preparation in Example 1) (reaction temperature, time: 94 ° C, denaturation for 5 minutes; then denaturation at 94 ° C 30 Seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute, amplification for 35 cycles; finally at 72 ° C for 10 minutes), the full-length ORF fragment of VSTM1-V2 cDNA was obtained, and then BamH I and The PCR product was digested with Sma I, and the prokaryotic expression vector pGEX4T-l was digested with BamH I and Sma I. The digested VSTM1-V2 cDNA gene fragment was ligated with the vector at 16 ° C for 8 hours to transform Escherichia coli. DH5a, transformants were grown on LB plate medium containing ampicillin, selected for growth, plasmids were extracted, PCR was identified, positive clones were selected, and the correct insert sequence of VSTM1-V2 cDNA was selected by sequencing (ibid.) The plasmid was named pGEX4T-l-VSTMl-v2. The expressed recombinant protein is GST-VSTM1-v2, which means that the N-terminus of the protein carries a GST tag, and the GST tag and the VSTM1-V2 target protein have a thrombin cleavage site, which facilitates digestion and purification. The pET-32a-c(+)-VSTMl-v2 clone construction process was consistent with pGEX4T-l-VSTMl-v2. The recombinant protein expressed by pET-32a-c(+)-VSTMl-v2 is Trx-His-S-VSTMl-v2, that is, the N-terminus of the protein carries three tags of Trx, His and S, and the His tag and the S tag There is a thrombin cleavage site between the S-tag and the VSTM1-V2 target protein, which has a restriction enzyme site for enterokinase, which facilitates more selective digestion and purification.

 2. Recombination VSTM1-V2 protein expression and purification

 (1) pGEX4T-l-VSTMl-v2

 The prokaryotic expression plasmid pGEX4T-l-VSTMl-v2 was transformed into E. coli BL21 (commercially available from companies such as TakaRa), grown on transformant LB plate medium (Amp resistant), and the toothpick single colony was attached to 5 ml LB (Amp resistant). (3), 3TC, 300rpm, overnight culture, then transferred to 100ml 2><YP (including Αιηρ Ι ΙΟμΙ) at 37 °C, 37 °C, 300 rpm, when the OD value is 0.7~0.8, add IPTG (final) Concentration is 0.6 mM), 22. C, 300 rpm, induced expression for 6 hours.

 Firstly, the bacterial solution was firstly identified by lml induction, centrifuged at 8000 rpm for 10 minutes, and the cells were centrifuged in 200 μl water, 5 times (400w, 10s ultrasound, 10s interval), 12000rpm, 4°C, 5min centrifugation. ΙΟΟμΙ supernatant, SDS-PAGE electrophoresis.

Then a large amount of purification was performed, and the induced GST-VSTM1-V2 engineering bacteria were collected and washed once with PBS at 8000 rpm. The heart was removed for 10 minutes, and the pellet was discarded, and the suspension # (PBS 10 ml/100 ml medium) was resuspended in PBS. Ultrasonic lysate 90 times (400w, 10s ultrasound, 10s interval, ice bath), 12000rpm, 4 °C, 20min centrifugation, discard the precipitate, leave the supernatant, and clear the 0.45um filter. Take lml Glutathione-Sepharose4B column, first wash the preservation solution in the column with deionized water, then equilibrate the column with PBS, add the filtered ultrasonic supernatant to the balanced column, and control the flow rate to about 3ml/min. After loading, rinse the column with PBS to the baseline. Close the outlet valve under the column, prepare thrombin into 40units/ml PBS solution, force the column to the column, and completely immerse the column in the enzyme solution, and let it stand for 1 hour at room temperature. Open the liquid outlet valve, collect the enzyme digestion solution, wash the column with 2ml PBS, and wash the column solution together with the previously collected enzyme digestion solution, and place it in the dialysis bag with pre-cooled lxPBS (pH 7.4) 4 °C. Dialysis twice, each time for 3 hours, then use PEG-2000 to concentrate protein to 500μ1 at 4 °C, aspirate the protein, sterilize at 4 °C, centrifuge at 18000g for 20 minutes, and take the supernatant and store at -80 °C. use.

 (2) pET-32a-c(+)-VSTMl-v2

 The prokaryotic expression plasmid pET-32a-c(+)-VSTMl-v2 was transformed into E. coli BL21, and the transformant LB plate medium (Amp resistance) was grown, and the toothpick single colony was ligated into 5 ml LB (Amp resistant), 37 °C, 300 rpm, overnight culture, then transferred to 100 ml LB (containing Αιηρ Ι ΙΟμΙ) at 37 °C, at 37 °C, 300 rpm, when the OD value is 0.7~0.8, IPTG (final concentration is 0.6 mM), Expression was induced for 6 hours at 22 °C, 300 rpm.

 Firstly, the bacterial solution was firstly identified by lml induction, centrifuged at 8000 rpm for 10 minutes, and the cells were centrifuged in 200 μl water, 5 times (400w, 10s ultrasound, 10s interval), 12000rpm, 4°C, 5min centrifugation. ΙΟΟμΙ supernatant, SDS-PAGE electrophoresis.

 Then, a large amount of purified Trx-His-S-VSTM1-v2 engineering bacteria was taken, washed once with PBS, centrifuged at 8000 rpm for 10 minutes, discarded, and the pellet was resuspended in PBS (PBS 20 ml/100 ml culture) base). Ultrasonic lysis bacteria 90 times (400w, 10s ultrasound, 10s interval, ice bath), 12000rpm, 4 °C, 20min centrifugation, discard the precipitate, leave the supernatant, and clear the 0.45um filter.

Purify the supernatant after treatment with M ²⁺ column: firstly filter the supernatant through a 0.45 μm filter, add imidazole (10 mM) / aCl (200 mM) to the supernatant, and then combine with the column, the flow rate is controlled at 10 drops. Every minute, use the equilibration buffer [imidazole (20 mM) / NaCl (200 mM) dissolved in lxPBS (pH 7.4)] to wash the column to wash away the unbound heteroprotein, and then elute to the baseline, elute The liquid [imidazole (500 mM) / NaCl (200 mM) dissolved in l x PBS (pH 7.4)] eluted the bound protein until the spectrophotometer measurement no longer decreased, collecting the eluate in the dialysis bag, using pre-cooled lxPBS ( PH7.4) Dialysis twice at 4 °C for 3 hours, then use PEG-2000 to concentrate protein to 500μ1 at 4 °C, aspirate the protein, sterilize at 4 °C, centrifuge at 18000g for 20 minutes, and take the supernatant. Store at -80 ° C until use.

 The two recombinant proteins were quantified by the BCA method (according to the BCATM Protein Assay Kit (Pierce, 23227) instructions), and a portion of the samples were subjected to SDS-PAGE to identify the purity of the purified VSTMl-v2 protein.

 Figure 3 A shows the purification of purified GST-VSTM1-v2 recombinant protein by SDS-PAGE and mass spectrometry.

(GST-rVSTM1-v2, lane 1 in the figure) and SDS-PAGE results after thrombin digestion (rVSTM1-v2, lane 2 in the figure). Two bands of SDS-PAGE appeared after purification of GST-VSTM1-V2 protein, and all of them were analyzed by mass spectrometry; After enzymatic digestion, the recombinant VSTM1-V2 band was single, in line with the expected size, and the purity was high.

 Figure 3B shows the results of SDS-PAGE analysis of the purification of Trx-His-S-VSTM1-v2 recombinant protein. It can be seen that the recombinant protein Trx-His-S-VSTMl-v2 has a slightly lower purity after purification, but has reached the requirements of the body.

 Example 5 Preparation, Purification and Identification of VSTM1 Antibody

 1. Preparation of prokaryotic protein VSTM1 polyclonal antibody

Animals were immunized with the purified Trx-His-S-VSTM1- _V 2 prokaryotic protein prepared in Example 4. Adult male New Zealand rabbits were used for primary immunization. 300 μ _§ of the antigen was diluted to 1 ml with PBS and mixed well with an equal volume of Freund's complete adjuvant. Each of the two feet was subcutaneously injected at 0.25 ml, and the rest of the back was subcutaneously multiplied (6 points). )injection. Then, every 3 weeks, the immunization was boosted once, and the amount was the same as before, and all the backs were injected subcutaneously (8 points). The titer of rabbit ear vein blood samples was taken 10 days after the third booster immunization. After the titer reached 1:10 ⁵ or more, the rabbits were sacrificed and blood was taken to obtain serum.

 An affinity chromatography column prepared by CNBR-activated Sepharose 4B coupled with GST-VSTM1-v2 prokaryotic protein. Then, the rabbit anti-VSTM1 anti-VSTM1 obtained above was spin-mixed at 4 ° C overnight, and the passage was taken for use, and the column was washed with PBS. An additional 0.1 M glycine buffer was added to the lake (pH 2.4), and the eluate was placed in a collection tube pre-added to 3 M Tris (pH 9.0). The intracellular antibody concentration was then measured using a microtiter plate. The specific antibody obtained was purified and dialyzed against a large volume of pH 7.4 PBS at 4 ° C and replaced three times with an interval of 8 hours. The protein was then concentrated to 1 ml with polyethylene glycol at 4 °C.

 Overexpression of VSTMl-v2 in HEK 293T cells (see Example 6 for details), antibody specificity was identified by Western blot, and the results are shown in Figure 4, indicating that this antibody specifically recognizes the exogenously expressed VSTM1-V2 protein.

 2. Preparation of VSTM1 polyclonal antibody by synthetic peptide

 In this example, according to the biological analysis of VSTM1-V2, the N-terminal amino acid sequence of VSTM1-V2 protein was selected: SEQ ID NO: 4, 17-32, and the 62-81 acid sequence shown in positions 62-81 (See the rectangular box in Figure 1, the two sequences are hydrophilic, antigenic and surface exposed, and no glycosylation modification), and they are returned to the protein database for matching, verifying Polypeptide synthesis after specificity (peptide was commissioned by Hangzhou Zhongpept Biochemical Co., Ltd.). The purity of the peptide is required to be greater than 75%, and some of the polypeptides are coupled to KLH.

Then, the antibody is prepared, and the immunized animal is selected from adult male New Zealand rabbits, two polypeptides which are coupled with KLH and the like, and the rabbits are immunized four times to prepare polyclonal antibodies. Initial immunization 300 μ _§ of the mixed polypeptide was diluted to 1 ml with PBS, mixed well with an equal volume of Freund's complete adjuvant, subcutaneously injected at 0.25 ml each of the two feet, and the other back was injected subcutaneously (6 points). Then, every 3 weeks, the immunization was boosted once, and the amount was the same as before, and all the backs were injected subcutaneously (8 points). The titer of rabbit ear vein blood samples was taken 10 days after the third booster immunization. After the titer reached 1:10 ⁵ or more, the rabbits were sacrificed and blood was taken to obtain serum. The serum was purified by CNBR coupled with VSTMl-v2 N-terminal polypeptide. The specificity of the antibody by Western blot (see Figure 5) showed that the antibody specifically recognized the exogenously expressed protein, thus obtaining a VSTM1-specific polyclonal antibody.

 Example 6. VSTM1-V2 3⁄41 secreted protein secreted by the classical pathway

Bioinformatics suggests that VSTM1-V2 is a secreted protein, which is verified by specific experiments in this example. 1. Cell culture, transfection: HEK 293T cells were transfected with plasmid pcDB-VSTMl-v2-myc-his.

HEK293T cells are presented to Professor T. Matsuda of Japan (also commercially available as HEK293T cells from ATCC), using DMEM (Dulbecco's modified Eagle's medium, 4.5 g/L glucose, 4 mM L-) containing 10% fetal bovine serum (FBS). Glutamine fiber, lOOU/ml penicillin, 100 _μ§ /ιη1 streptomycin).

The target gene pcDB-VSTMl-v2-myc-his eukaryotic expression plasmid was transfected using Vigofect cation transfection. The specific steps are as follows: (1) Cell culture: HEK 293T cells (3.0×10 ⁵ ) were plated in 10 cm culture dish with DMEM medium containing 10% fetal bovine serum at 5% CO ₂ , 37 ° C. Also after 2μ1 VigoFect diluted with 100μ1 ΡΒ8, slowly mixed, allowed to stand at room temperature for 5 min; target plasmid was diluted with 5μ _§ lOO ^ PBS, mixed slowly:; culture incubator for 24 hours (2) preparation of the complex DNA- Vigofect , slowly mix with diluted DNA, leave it at room temperature for 15 minutes to form DNA-VigoFect complex; (3) Transfection: Slowly drop DNA-VigoFect complex into cell culture plate (200μ1/well), shake gently; Incubate in an incubator at %C0 ₂ at 37 °C.

 After 24 hours of transfection, 10 mg/ml BFA (Brefeldin-A, brefeldin A) and DMSO (used as a negative control) were added to the cell culture supernatant, and after 24 hours of cell treatment, the cell fusion rate was about At 90%, the cells of each experimental group and the cell culture supernatant were collected for Western blot analysis.

 2. Obtain cell culture supernatant and extract cell protein and detect the overexpression of VSTM1 protein by Western blot.

Collection of 293T cell culture supernatant: After 48 hours of cell transfection, the cell culture supernatants of each experimental group were collected, centrifuged at 2000 g for 10 minutes at 4 ° C, discarded, and centrifuged at 15000 g for 15 minutes at 4 ° C, then The mixture was concentrated under vacuum at 37 ° C for 1 hour, and the treated cell culture supernatant was taken.

 Extraction of total protein from 293T cells: The supernatanted cells were placed on ice, washed twice with ice-cold l xPBS, and the cells were blown off with ice-cold l x PBS, and the cells were collected into 1.5 mL centrifuge tubes. Centrifuge at 2000g for 5 minutes at 4 °C. The supernatant was removed, and RIPA cell lysate (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, ImM EGTA, 1% Triton X-100, protease inhibitor Cocktail) was added to the pellet and placed on ice for 30 minutes, 4°. C, centrifuge at 15000g for 15 minutes, transfer the supernatant to a new tube, and store at -80 °C for later use.

 Protein quantification: Cell-extracted proteins were quantified according to the BCATM Protein Assay Kit (Pierce, 23227) specification.

Western blot: Take 30μ of each cell protein. _§ 40μ1 of each cell culture supernatant, add protein loading buffer (Beijing Baosai Biotechnology Co., Ltd.), and cook in a water bath at 100 °C for 5 minutes. 12.5% PAGE electrophoresis, lOOmV electrospinning for 1 hour, TBST solution equilibration, blocking with 5% milk at room temperature for 2 hours, respectively, adding VSTM1 polyclonal antibody (1: 1000) prepared by synthesizing polypeptide in Example 5, monoclonal anti-β-actin Antibody (Sigma, 1:3000) as primary antibody, 4 overnight, wash membrane 3 times with TBST for 10 minutes each time; then add the corresponding IRDyeTM 800 labeled secondary antibody (1: 10000), avoiding light at room temperature 1 Hours; Wash the membrane thoroughly 3 times with TBST for 10 minutes each time; Finally, the signal was detected at 800 nm using the Odyssey Infrared Imager.

The results of Western blot analysis are shown in Figure 5, in the culture supernatant of 293T cells overexpressing VSTMl-v2, VSTMl-v2 can be detected, but its protein molecular weight is too large, possibly due to modifications such as glycosylation. After the addition of the inhibitor BFA of the classical secretory pathway (endoplasmic reticulum Golgi pathway), VSTM1-V2 secretion was significantly reduced, demonstrating that VSTMl-v2 is a secreted protein secreted by the classical pathway.

 Example 7. Eukaryotic cells VSTM1-V2 secreted protein and purification

HEK 293T cells were plated in 10 cm culture dishes in DMEM medium containing 10% fetal bovine serum, and cultured in a 5% CO ₂ , 37 ° C incubator for 24 hours. The gene of interest pcDB-VSTMl-v2-myc-his eukaryotic expression plasmid was transfected using Vigofect cation transfection (see Example 6). After 6 hours of transfection, the cells were washed once with normal temperature lxPBS, replaced with fresh serum-free medium (but with low protein culture cytokines), and cultured in a 5% CO ₂ , 37 ° C incubator. After 48 hours, the cell culture supernatant was collected and centrifuged at 2000 g for 10 minutes at 4 ° C. The purpose was to remove the cells of the cell culture supernatant, discard the pellet, and centrifuge at 20 °C at 18000 g at 4 ° C to remove small particles in the supernatant. Substance, the treated cell culture supernatant was taken. The above-mentioned cell culture supernatant was purified by Ni ² + column: The specific method was the same as in Example 4, and the supernatant after the treatment was stored at -80 ° C until use. Take 5μ1 protein and quantify BCA protein by BCA method. Take some samples (30μ _{§ for} each group of cell proteins), add protein loading buffer, boil in 100 °C water bath for 5 minutes, perform SDS-PAGE and western blot to identify Purity of VSTMl-v2 secreted protein purification.

 The results are shown in Figure 6. The purity of the purified protein of VSTM1-V2 is high. Example 8. Purification of eukaryotic cells Glycosylation assay of VSTM1-V2 secreted protein

 3χ30μ1 of the eukaryotic cells prepared in Example 7 were purified VSTM1-V2 protein, added with 0.1% SDS and 50 mM β-mercaptoethanol, denatured at 95 °C for 5 minutes, then added with 1% ΝΡ-40, protease inhibitor cocktail, respectively. N-glycosidase F, 0-glycosidase and double distilled water were digested at 37 °C for 2 hours, then the protein-loading buffer was added to terminate the digestion reaction, and the glycosylation of VSTM1-V2 was detected by western blot.

 The results are shown in Fig. 7. Compared with the control (Ball 1) in which double distilled water was added, the VSTM1-V2 band (lane 2) was significantly smaller and denser after the addition of Ν-glucosidase F, and 0-glycosidase was added. There was no significant change in the posterior (lane 3), suggesting that the VSTM1-V2 secreted protein has a glycosylation modification without a glycosylation modification. Real deletion 9. Detection of the effect of VSTM1-V2 on the activation and differentiation of CD4+ T cells

 1. Detection of the effect of VSTM1-V2 secreted protein on the expression of cytokines in CD4+ T cells

PBMC (normal human peripheral blood mononuclear cells) was isolated from normal human peripheral blood by lymphocyte stratification, and CD4+ T lymphocytes were purified from positive beads, and the magnetic beads were purified to obtain CD4+T. The lymphocytes were cultured in a cell culture plate coated with anti-CD3 (^g/ml; clone 0KT3) and anti-CD28 (2 μ§/ηύ _; clone 15E8) antibody, and the density was 1.5χ10 ⁶ finely expanded ml, divided into seven. Group, respectively, add PBS, eukaryotic cell VSTMl-v2 secreted protein (lng, 10ng, lOOng/ml), heat inactivated eukaryotic cells VSTMl-v2 secreted protein (100ng/ml), prokaryotic cell VSTMl-v2 protein (100ng/ Ml, GST-VSTMl-v2 recombinant protein removed GST tag) and its heat inactivated protein, the above seven groups were defined as PBS, VSTMl-v2-l VSTMl-v2-10 VSTMl-v2-100 AVSTMl-v2-100 rVSTMl- V2 and ΔrVSTMl-v2.

At 37 ° C, 5% CO ₂ was cultured for 69 hours, BFA was added to inhibit the secretion of cytokines, and cells were harvested at 72 hours by flow cytometry (FACS) to detect intracellular IFN-y IL-4, IL-17A, etc. Changes in cytokines. Intracellular Molecular Detection: Different types of immune cells were harvested, washed twice with cold PBS/0.1% BSA, first fixed on ice with 3% polyformic acid for 30 min; then incubated with 0.1% Triton X-100 for 30 min at room temperature, 1500 rpm Centrifuge for 5 min. The cells were resuspended by adding ΙΟΟμΙ blocking solution (PBS/10% normal goat serum) and blocked at room temperature for 30 minutes. Subsequently, PE-labeled IFN-γ antibody (BD), FITC-labeled IL-4 antibody (BD), APC-labeled IL-17A antibody (R&D) were added, and incubated at 4 ° C for 40 min in the dark _{; the} corresponding IgG was used as a negative control. After finally washing the paint twice with PBS/0.1% BSA, the cells were collected by flow cytometry, and the results were analyzed by the shed Cellquest software.

 The results of the assay are shown in Figure 8. There was no significant change in IFN-γ and IL-4 (Picture A, B), whereas eukaryotic and prokaryotic proteins

Both VSTM1-V2 can significantly increase the number of cells secreted by IL-17A (Picture C). Since IL-17A is mainly secreted by TM7 cells in CD4+ T cells, the results of this example suggest that VSTM1-V2 may affect the differentiation of TM7 cells.

 2. Detection of the effect of VSTM1-V2 secreted protein on the differentiation of TM7 cells

Similarly, PBMC (normal human peripheral blood mononuclear cells) was isolated from normal human peripheral blood by lymphocyte stratification, and then CD4 ⁺ T lymphocytes were purified from positively sorted magnetic beads, and small cells were added to the purified cells. Mouse anti-human CD45RO antibody (BD) and rat anti-mouse IgG2a positive sorting magnetic beads (pynal Biotech) were used to remove CD45RO+ cells, and the remaining cells were CD45RA+ Naive T lymphocytes. The purity of the sorted CD4 ⁺ Naive T lymphocytes was identified by flow cytometry using fluorescently labeled anti-CD45RA, CD45RO and CD4 antibodies.

 Naive T lymphocytes were cultured in cell culture plates coated with anti-CD3 (^g/ml; clone OKT3) and anti-CD28 (2 μ§/πύ·, clone 15E8) antibodies, and recombinant human IL- was added to the culture system. Ιβ (50 ng/ml), recombinant human IL-6 (50 ng/ml), recombinant human IL-23 (50 ng/ml), recombinant human TNF-a (10 ng/ml), recombinant human TGF-βΙ ( 5 ng/ml), anti-human IL-4 antibody (5 μ^ιηΐ) and anti-human IF-γ (5 μ^ιηΐ) antibodies. Four days later, the cells were exchanged for anti-CD3 and anti-CD28 antibodies, recombinant human IL-2 (5n^ml) was added, and the remaining cytokines and antibodies were unchanged, and culture was continued for three days. In the process of TM7 cell differentiation, PBS, eukaryotic cell VSTM1-V2 secreted protein (lng/ml, lOng/mk 100ng/ml) were added to detect the effect of VSTM1-V2 secreted protein on the differentiation of TM7 cells.

 After 7 days of differentiation, TM7 cells were stimulated with PMA (100 ng/ml) and ionomycin (ΙμΜ) for 6 hours, in which BFA was added to inhibit cytokine secretion 3 hours later, and IL-17A levels in the cells were detected by FACS. Results Referring to the picture in Figure 9, VSTM1-V2 promoted IL-17A+TM7 cell proliferation.

 In addition, 7 days after the differentiation of TM7 cells, the cell culture supernatant was harvested, and the secretion of cytokine IL-17A was detected by ELISA. The results are shown in Figure B in Figure 9, and it can be seen that VSTM1-V2 promotes IL-17A secretion.

Further, RA of each of the above groups of cells was extracted, and the change in IL-17A transcription level was detected by real time PCR method (Real-time PCR using SYBR Green method and LightCycler instrument). The results are shown in Figure C in Figure 9, and consistent with the FACS results, VSTMl-v 2 can express IL-17A expression in Thl7 cells. In addition, cell proliferation was detected by [ ³ H]-TdR incorporation assay. Specific experimental procedures: CD4+ Naive T lymphocytes were isolated and suspended in 10% FBS RPMI 1640 to adjust the cell concentration to 2>< 10 ⁶ /ml. . The 96-well flat-bottomed plate coated with anti-CD3 and CD28 antibody was added, ΙΟΟμΙ/well, and the cultured bovine was the same as the induced ΤΜ7 cell differentiation system, and APBS or eukaryotic cell VSTM1-V2 secreted protein was added, and 53 replicate wells were set in each group. After 7 days of culture, [ ³ H]-TdR was added at a concentration of Ιμθ/ιηΐ, and after continuing to culture for 18 hours, the cells were collected on a 96-well Filtermat, and the incorporation of [ ³ H]-TdR was measured by McroBeta Windows Workstation (Wallac). The [ ³ H]-TdR incorporation assay revealed that VSTM1-V2 secreted protein (1ng/ml) promoted the proliferation of TM7 cells. The results are shown in Figure D in Figure 9.

 These results indicate that VSTM1-V2 can promote the differentiation of Thl7 cells in vitro. Example 10: Detection of the effect of VSTM1-V2 on the activation and killing function of CD8+ T cells

First, PBMC (normal human peripheral blood mononuclear cells) was isolated from normal human peripheral blood by lymphocyte stratification, and then purified from CD8+ T lymphocytes by positive sorting magnetic beads, and cultured with anti-CD3 ( 1 _{μ §} /ιη1; clone ΟΚΤ3 ) and anti-CD28 (2 μ^ιηΐ; clone 15E8) antibody-coated cell culture plates, with a density of 1.5 χ 10 ⁶ finely swelled ml, divided into two groups, respectively, adding PBS and eukaryotic cells VSTM1-V2 secreted protein (10 ng/ml).

After 37 hours of incubation at 37 ° C, 5% CO ₂ cattle, BFA was added to inhibit the secretion of cytokines, and cells were harvested for 72 hours to detect changes in intracellular cytokine IFN-γ by FACS. The results are shown in Figure A in Figure 10. It was found that VSTMl-v2 can significantly promote the expression of IFN-γ in CD8+ T cells.

When detecting the function of CD8+ T cells killing K562 cells, the CD8+ T lymphocytes purified by magnetic beads were cultured in cells cultured with anti-CD3 (^g/ml) and anti-CD28 (2 μ^ιηΐ) antibodies. The plate has a density of 1.5><10 ⁶ finely swelled ml, the cells are co-cultured for six days, and the fresh medium is changed in the middle. After six days, CD8+ T cells are co-incubated with K562 cells at a ratio of 9:1, and anti-CD3 is added at the same time. ( ^g / ml) and anti-CD28 (2 g / ml) antibody, additionally added PBS or eukaryotic cell VSTMl-v2 secreted protein (10 ng / ml), 24 hours after the observation of CD8 + T fine K562 cells under light microscope The killing effect and the apoptosis of K562 cells were detected by flow cytometry Annexin V-FITC/PI double staining, which reflected the effect of VSTMl-v2 secreted protein on the function of CD8+ T cells to kill K562 cells.

The specific procedure for detecting apoptosis by flow cytometry is as follows: Harvest the cells and prepare a single cell suspension. After washing the pre-cooled PBS twice, change the binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM MgC12). , 5mM KC1, 2.5mM CaC12) Wash the cells once, add FI C-Annexin V to a final concentration of 0.5 (og/ml, incubate at 4 °C for 30 minutes, add _propidium iodide to a final concentration of 1 _μ§ /ιη1, upflow cytometry , programmed cell death was detected by 488 nm argon excitation.

 The detection results are shown in Figure B, picture B and picture C. It was found that VSTM1-V2 can significantly promote the aggregation of CD8+ T cells and K562 cells and the apoptosis of K562 cells, suggesting that VSTMl-v2 can promote CD8+ T cell pairs. The killing effect of K562 cells. Example 11. Identification and analysis of VSTM1-V2 transgenic mice

 1. Identification of VSTM1-V2 transgenic mice

In order to study the in vivo function of VSTM1-V2, the present invention was commissioned by the Institute of Experimental Animals of the Chinese Academy of Medical Sciences. VSTM1-V2 transgenic mice. The VSTM1-V2 transgenic mice were first identified for genome level using PCR, as shown in panel A of Figure 11. Further identification at the protein level, since VSTM1-V2 can be secreted into the peripheral blood, VSTM1-v2 in the serum is detected by indirect ELISA, and the results are shown in Figure B, picture B, genomic-positive mouse serum. The VSTM1-V2 was higher than the wild type, suggesting that the integrated VSTM1-V2 plasmid was successfully expressed, but the expression level of VSTM1-V2 was different, which may be due to the different copy number of VSTM1-V2. Albumin and IgG in serum were removed, purified by M-SepharoseTM 6 FastFlow column, and identified by SDS-PAGE and Western blot. The results are shown in Figure C, picture C, compared with wild type mice, in VSTMl- The VSTM1-V2 specific band was observed in the serum of v2 transgenic mice, and it was consistent with the VSTM1-V2 protein expressed by purified eukaryotic cells. It also showed three bands, suggesting that VSTM1-V2 has also undergone processing modification in mice, which may be Mature active form.

 2. Appearance of major organs and tissue HE staining in VSTM1-V2 transgenic mice

 After obtaining VSTM1-V2 positive mice, the phenotype was first studied. There was no significant change in the appearance of the VSTMl-vl transgenic mice and the appearance of each major organ compared to wild-type mice. The main tissues were taken for fixation, sectioning, and HE staining. The results are shown in Figure 12. Compared with wild-type mice, male VSTM1-V2 transgenic mice had many mature spermatozoa and sperm cells in the testicular tissue seminiferous tubules. In addition, it was found that the fertility of VSTM1-V2 transgenic mice was stronger than that of wild-type and VSTM1-vl transgenic mice, suggesting that VSTM1-V2 may have a role in promoting sperm development. Other tissues including the immune system showed no significant changes.

 3. Blood routine analysis

 Blood was drawn from the mice and anticoagulated for routine blood analysis. The results are shown in Figure 13. Compared with wild-type mice, VSTM1-V2 transgenic mice showed an increase in the absolute number of granulocytes, a slight increase in monocytes, and the number of lymphocytes. It is basically unchanged, resulting in an increase in the ratio of granulocytes and monocytes, and a decrease in the proportion of corresponding lymphocytes, suggesting that VSTM1-V2 may have an effect on the production and survival of granulocytes and monocytes. There is no significant difference in other aspects.

 4. Detection of serum cytokine levels in VSTM1-V2 transgenic mice

 The concentrations of IFN, IL-4, IL-17A and other cytokines in serum were measured by ELISA. As shown in Figure 14, the levels of IFN-γ and IL-17A in VSTMl-v2 transgenic mice were higher than those in wild-type mice. IL-4 was not detected due to its low content.

 5. Analysis of spleen cell subsets and cytokines in VSTM1-V2 transgenic mice

The spleen cells were obtained by grinding the mouse spleen and immunofluorescence staining. The results are shown in Figure A, picture A, CD4+, Treg (CD4+CD25+FoxP3+) of VSTM1-V2 transgenic mice compared with wild type mice. The proportion of several immune cell subpopulations such as CD8+, CDl lc+ and CD16/32+ did not change significantly. Another part of the spleen cells were stimulated with PMA and ionomycin for 4 hours to detect the expression and secretion of related cytokines. The FACS results are shown in panel B of Figure 15. Compared with wild-type mice, VSTM1-V2 transgenic mice have increased IL-17A+ cells and increased mean fluorescence intensity; ΙΡΝ-γ+ cells are also more, but There was no significant difference in mean fluorescence intensity; there was no significant difference in the number of IL-4+ cells and their mean fluorescence intensity. different. The spleen cell culture supernatant ELISA results (see picture C in Figure 15) were consistent with the cytokine test results in serum, but the IFN-Y and IL-17A levels in VSTM1-V2 transgenic mice were significantly higher than wild type. Mice, IL-4 failed to detect.

 6. VSTM1-V2 promotes and aggravates the experimental autoimmune encephalomyelitis in mice. The pathogenesis and progression of EAE Research reports Thl7 cells are involved in the development and progression of autoimmune diseases and play a leading role in the mouse EAE model. To further investigate the effect of VSTM1-V2 on TM7 cells in vivo, the present invention established an EAE model using VSTM1-V2 transgenic mice. The results showed that VSTM1-V2 transgenic mice were more sensitive to EAE than wild-type mice, with an onset 2 days earlier, and the condition may be more severe (see Figure A, picture A and picture B), proving that VSTM1-V2 does Participated in the occurrence and development of the autoimmune disease model EAE. Picture C in Figure 16 shows the highest clinical score of a single mouse during the observation period. The average highest clinical score of VSTM1-V2 transgenic mice was higher than that of wild-type mice, but there was no statistically significant difference due to the large individual differences. Figure D shows HE staining of spinal cord sections of diseased mice, showing infiltrating lymphocytes. After the observation period of the 26th day of the EAE model was established, the mice were sacrificed to take spleen cells, and the proportion of each immune cell subpopulation was detected by FACS. As a result, it was found (see picture E in Fig. 16) that the proportion of TM4 cells of CD4+IL-17A+ was Significantly increased, the mean fluorescence intensity also increased, while the Th1 and Th2 cell subsets did not change significantly, suggesting that VSTM1-V2 may accelerate the onset of EAE by promoting TM7 cell development. Example 12, VSTM1-V2 in the serum of patients with autoimmune diseases;

 In order to clarify the role of VSTM1-V2 in human autoimmune diseases, the present invention also detects the expression of VSTM1-V2 in the serum of autoimmune patients. As shown in Figure 17, in patients with rheumatoid arthritis (RA), serum VSTM1-V2 levels were significantly higher in some patients than in healthy controls, suggesting that VSTM1-V2 may be involved in the development of autoimmune diseases. The above examples demonstrate that VSTM1-V2 acts as a secreted protein that mediates the differentiation and regulation of Th cells in vitro.

The function of CD8+ T lymphocytes, in vivo, may serve as a cytokine that plays an important role in the immune system. VSTM1-V2 protein and VSTM1 antibody have potential clinical application value in anti-infective, immune-enhancing, anti-autoimmune diseases and anti-tumor immunity.

Claims

Claim 1. A protein or an immunological fragment thereof as shown in (a) or (b) below:  (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an immunological fragment thereof; or  (b) a protein derived from (a) or an immunological fragment thereof which has been substituted, deleted or added with one or several amino acids in (a) a defined amino acid sequence and which has the same function as (a);  The immunological fragment is preferably a polypeptide consisting of the amino acid sequence shown in positions 17 to 32 or 62 to 81 of SEQ ID NO: 4, or the amino acid sequence shown in positions 17 to 205 of SEQ ID NO: 4. Protein. A polynucleotide sequence encoding the protein of claim 1 or an immunological fragment thereof; wherein the polynucleotide sequence is preferably the polynucleotide sequence of SEQ ID NO: 3. 3. A genetically engineered vector comprising the polynucleotide sequence of claim 2. 4. The genetically engineered vector of claim 3, wherein the vector is a plasmid. A host cell which is transformed, transfected or transduced with the genetically engineered vector of claim 3 or 4. 6. A protein according to claim 1 or an immunological fragment thereof or an antagonist of the polynucleotide sequence of claim 2.  7. An antagonist according to claim 6 which is an antibody, antisense RNA or small interfering RNA. The use of the protein of claim 1 or an immunological fragment thereof or the polynucleotide sequence of claim 2 for promoting Thl7 differentiation and/or killing function of CD8+ T lymphocytes. 9. The protein of claim 1 or an immunological fragment thereof, or the polynucleotide sequence of claim 2, or the antagonist of claim 6 or 7, in the preparation of a medicament for the prevention and/or treatment of immunity The use of a pharmaceutical composition for a disease. 10. The use according to claim 9, wherein the protein is a protein consisting of the amino acid sequence shown in positions 17 to 205 of SEQ ID NO: 4; the polynucleotide sequence is SEQ ID NO: 3 The polynucleotide sequence shown; the antagonist is a monoclonal antibody or a polyclonal antibody; and the immune-related disease is an infectious disease, an autoimmune disease or a tumor. The use according to claim 9 or 10, wherein said polynucleotide sequence is contained in a vector. 12. The use according to claim 11, wherein the vector is a plasmid. A composition for preventing and/or treating an immune-related disease, the composition comprising the protein of claim 1 or an immunological fragment thereof, or the polynucleotide sequence of claim 2, or the claims The vector of 3 or 4, or the host cell of claim 5, or the antagonist of claim 6 or 7; and one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers. The use of the protein of claim 1 or an immunological fragment thereof or the polynucleotide sequence of claim 2 for the preparation of a composition for the auxiliary diagnosis and prognosis of an immune-related disease. 15. The use according to claim 14, wherein the reagent is an antibody, an antisense RNA or a small interference