WO2011038689A1 - Nouvelle cytokine humaine vstm1-v2 et son utilisation - Google Patents

Nouvelle cytokine humaine vstm1-v2 et son utilisation Download PDF

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WO2011038689A1
WO2011038689A1 PCT/CN2010/077499 CN2010077499W WO2011038689A1 WO 2011038689 A1 WO2011038689 A1 WO 2011038689A1 CN 2010077499 W CN2010077499 W CN 2010077499W WO 2011038689 A1 WO2011038689 A1 WO 2011038689A1
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protein
vstm1
cells
polynucleotide sequence
immunological fragment
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PCT/CN2010/077499
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English (en)
Chinese (zh)
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马大龙
韩文玲
郭晓欢
王平章
李婷
黄晶
郭金海
付伟伟
张岩飞
石太平
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • 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.
  • 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.
  • IL Interleukin
  • CSF Colony-Stimulating Factor
  • IFN Interferon
  • TNF Tumor-Necrosis Factor
  • CK Chemokine
  • GF Growth Factor
  • 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).
  • ⁇ 7 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.
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • asthma asthma
  • lupus IL-17 expression in transplant rejection
  • 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).
  • UC ulcerative colitis
  • CD Crohn's disease
  • 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- ⁇ , 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.
  • IL-23 plays a very important role in maintaining differentiation into TM7 cells; inflammatory cells such as TNF-o IL- ⁇ 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- ⁇ and IFN- ⁇ secreted, which in turn kill target cells.
  • recombinant cytokines or recombinant soluble receptors produced by genetic engineering technology and therapeutic antibodies are used to treat tumors,
  • 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).
  • cytokine drugs include interferon alpha, beta, gamma, Epo, GM-CSF, G-CSF, IL-2 and the like.
  • 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
  • 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:
  • 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.
  • 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;
  • 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 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • lane 1 is the supernatant of bacterial lysis
  • 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.
  • lane 1 was a negative control group transfected with empty vector
  • 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- ⁇ (; 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.
  • 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.
  • picture A shows the results of FACS detection of intracellular cytokine IFN- ⁇
  • 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.
  • 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.
  • Figure 12 shows the results of HE staining of tissues of wild type and VSTM1-V2 transgenic mice.
  • 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.
  • 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.
  • 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.
  • LY lymphocyte
  • MO monocyte
  • GR granulocyte
  • WT wild type mouse
  • n 4
  • TG VSTM1-V2 transgenic mouse
  • n 8.
  • Figure 14 shows elevated levels of IFN- ⁇ and IL-17A in the serum of VSTMl-v2 transgenic mice.
  • Figure 15 shows that VSTM1-V2 promotes the expression of IFN- ⁇ 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.
  • FIG 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.
  • Figure 17 shows the expression level of VSTM1-V2 in the serum of patients with rheumatoid arthritis. detailed description
  • 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).
  • VSTMl-v2 encodes a classical secretory protein, i.e., the VSTM1-V2 protein of the present invention.
  • the present invention provides the protein represented by the following (a) or (b) or an immunological fragment thereof:
  • 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.
  • 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.
  • 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)).
  • coding sequence CDS: nucleoside 12 to 629
  • 5' non-coding region nucleotides 1 to 11
  • 3' non-coding region nucleotide No. 630 ⁇ 640
  • an isolated nucleotide sequence which comprises only the coding sequence of the VSTM1 protein is more preferred.
  • 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.
  • 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).
  • 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.
  • 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.
  • the guanosine can be cloned into a vector and then obtained by replication in the vector.
  • 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 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.
  • such vectors should contain an appropriate promoter, ribosome binding site, terminator, and the like.
  • an appropriate leader sequence can be added upstream of the polypeptide coding sequence.
  • an enhancer sequence can be inserted into the vector. Selection of suitable vectors and promoters is well known to those skilled in the art.
  • vectors comprising the polynucleotides of the invention and suitable transcriptional and translational regulatory components are well known to those skilled in the art.
  • 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.
  • telomeres 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.
  • 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.
  • pcDT ⁇ 5-His-TOPO
  • 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.
  • 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.
  • expression vectors 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 ⁇ 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.
  • prokaryotic hosts such as Escherichia coli, Bacillus, Streptomyces, etc.
  • eukaryotic hosts such as: Saccharomyces, Aspergillus, insect cells, various fly s2 and grass ⁇ S ⁇
  • plant cells Animal cells, such as CHO, COS (monkey kidney fibroblast cell line, Gluzman (Cell 23: 175, 1981)) or Bowes mel
  • 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.
  • MgCl 2 treatment An alternative is MgCl 2 treatment, which can also be treated by electroporation.
  • 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.
  • the promoter is induced by an appropriate method (e.g., variation or chemical trait induction), and then the culture is continued.
  • 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 .
  • nucleic acid fragments homologous to any portion of a polynucleotide of the invention.
  • 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
  • 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).
  • polyclonal antibodies can be immunized with the polypeptide protein of the present invention or an immunological fragment thereof , such as rabbits, mice, rats, etc.
  • the antigenic VSTM1 N-terminus SEQ ID NO: 4 amino acid sequences 17 to 32 and 62) ⁇ 81
  • VSTM1 prokaryotic protein amino acid sequence 17-205 of SEQ ID NO: 4
  • 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.
  • 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.
  • 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.
  • CD8+ T lymphocytes act as CTLs after activation, killing target cells and playing an important role in anti-tumor and anti-viral immunity.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 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.
  • 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.
  • 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.
  • RFLP Restriction fragment length polymorphism analysis
  • RT-PCR reverse transcription-polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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 £ ⁇ 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- ⁇ , 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.
  • 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.
  • i TRIzol invitrogen
  • R A granulocyte total R A
  • reverse transcriptTM kit CInvitrogen 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 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).
  • a human normal spleen tissue cDNA library (Clontech: Cat. No.
  • 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 50 ⁇ 1, which contains: human normal spleen/fetal liver tissue granulocyte cDNA template: 5 ⁇ 1 (5ng); Primer: outer forward primer, reverse primer final concentration 0.2 ⁇ ; dNTP: final concentration 200 ⁇ ; Taq DNA polymerization Enzyme: 2.5 U; lOxTaq DNA polymerase buffer: 5 ⁇ l; make up to 50 ⁇ l volume with double distilled water.
  • 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 ⁇ l, which contained: 50 ⁇ M diluted by a product: 5 ⁇ l (5 ng); The medial forward arches were 0.2 ⁇ each at the final concentration of the reverse primer; dNTP: 200 ⁇ at the final concentration; Taq DNA polymerase: 2.5 U; lOxTaq DNA polymerase buffer: 5 ⁇ l; 50 ⁇ l volume supplemented with double distilled water.
  • 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).
  • 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.
  • Fig. 1 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 1 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.
  • 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.
  • 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.
  • 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.
  • VSTM1-V2 also has a certain abundance in the liver, placenta and ovaries.
  • a eukaryotic expression plasmid containing VSTM1-V2 cDNA was constructed in this example: pcDNA3.
  • pcDNA3 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/myc
  • the VSTM1-V2 cDNA gene fragment after cleavage is ligated to the vector at 16 °C.
  • 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
  • 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.
  • 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 p
  • 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.
  • 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).
  • the bacterial solution was firstly identified by lml induction, centrifuged at 8000 rpm for 10 minutes, and the cells were centrifuged in 200 ⁇ l water, 5 times (400w, 10s ultrasound, 10s interval), 12000rpm, 4°C, 5min centrifugation. ⁇ supernatant, SDS-PAGE electrophoresis.
  • 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 ⁇ ⁇ ⁇ ) 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.
  • the bacterial solution was firstly identified by lml induction, centrifuged at 8000 rpm for 10 minutes, and the cells were centrifuged in 200 ⁇ l water, 5 times (400w, 10s ultrasound, 10s interval), 12000rpm, 4°C, 5min centrifugation. ⁇ supernatant, SDS-PAGE electrophoresis.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • Trx-His-S-VSTM1- V 2 prokaryotic protein prepared in Example 4.
  • Adult male New Zealand rabbits were used for primary immunization. 300 ⁇ ⁇ 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.
  • 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.
  • 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 ⁇ ⁇ 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 showed that the antibody specifically recognized the exogenously expressed protein, thus obtaining a VSTM1-specific polyclonal antibody.
  • Bioinformatics suggests that VSTM1-V2 is a secreted protein, which is verified by specific experiments in this example.
  • 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 ⁇ / ⁇ 1 streptomycin).
  • DMEM Dulbecco's modified Eagle's medium, 4.5 g/L glucose, 4 mM L-
  • FBS fetal bovine serum
  • Glutamine fiber lOOU/ml penicillin, 100 ⁇ / ⁇ 1 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.
  • Western blot Take 30 ⁇ of each cell protein. ⁇ 40 ⁇ 1 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.
  • protein loading buffer Beijing Baosai Biotechnology Co., Ltd.
  • 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 5 ⁇ 1 protein and quantify BCA protein by BCA method. Take some samples (30 ⁇ ⁇ 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.
  • 3 ⁇ 30 ⁇ 1 of the eukaryotic cells prepared in Example 7 were purified VSTM1-V2 protein, added with 0.1% SDS and 50 mM ⁇ -mercaptoethanol, denatured at 95 °C for 5 minutes, then added with 1% ⁇ -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.
  • 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 ⁇ -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
  • PBMC normal human peripheral blood mononuclear cells
  • 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 ⁇ / ⁇ ; clone 15E8) antibody, and the density was 1.5 ⁇ 10 6 finely expanded ml, divided into seven.
  • cytokines 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 ⁇ blocking solution (PBS/10% normal goat serum) and blocked at room temperature for 30 minutes.
  • ⁇ blocking solution PBS/10% normal goat serum
  • PE-labeled IFN- ⁇ 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.
  • 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.
  • PBMC normal human peripheral blood mononuclear cells
  • 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 ⁇ / ⁇ , clone 15E8) antibodies, and recombinant human IL- was added to the culture system.
  • ⁇ 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- ⁇ 5 ng/ml
  • anti-human IL-4 antibody 5 ⁇
  • anti-human IF- ⁇ 5 ⁇
  • 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.
  • TM7 cells were stimulated with PMA (100 ng/ml) and ionomycin ( ⁇ ) 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.
  • 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.
  • cell proliferation was detected by [ 3 H]-TdR incorporation assay.
  • CD4+ Naive T lymphocytes were isolated and suspended in 10% FBS RPMI 1640 to adjust the cell concentration to 2> ⁇ 10 6 /ml. .
  • 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
  • PBMC normal human peripheral blood mononuclear cells
  • the CD8+ T lymphocytes purified by magnetic beads were cultured in cells cultured with anti-CD3 ( ⁇ g/ml) and anti-CD28 (2 ⁇ ) 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.
  • 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 ⁇ / ⁇ 1, upflow cytometry , programmed cell death was detected by 488 nm argon excitation.
  • 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.
  • VSTM1-V2 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.
  • 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.
  • 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- ⁇ 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.
  • 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.
  • VSTM1-V2 transgenic mice Compared with wild-type mice, VSTM1-V2 transgenic mice have increased IL-17A+ cells and increased mean fluorescence intensity; ⁇ - ⁇ + 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.
  • VSTM1-V2 promotes and aggravates the experimental autoimmune encephalomyelitis in mice.
  • 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.
  • the present invention also detects the expression of VSTM1-V2 in the serum of autoimmune patients.
  • 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.
  • 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.

Abstract

La présente invention concerne une nouvelle cytokine humaine VSTM1-v2, son gène codant ou ses fragments d'immunité, l'utilisation de ladite protéine, dudit gène ou de ses fragments d'immunité pour favoriser la différentiation de Th17 et la fonction tueuse des lymphocytes T CD8+ et l'utilisation de ladite protéine, ledit gène ou de ses fragments d'immunité dans la préparation d'une composition pharmaceutique pour la prévention et/ou le traitement de maladies liées à l'immunité. La présente invention porte également sur des antagonistes de VSTM1-v2 tels que des anticorps monoclonaux ou des anticorps polyclonaux, sur un vecteur, une cellule hôte ou une composition comprenant VSTM1-v2 et sur un réactif pour tester VSTM1-v2 ou ses fragments d'immunité, et sur leur utilisation.
PCT/CN2010/077499 2009-09-29 2010-09-29 Nouvelle cytokine humaine vstm1-v2 et son utilisation WO2011038689A1 (fr)

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