Application of VSIG2 in the preparation of products for the treatment of autoimmune diseases

Abstract

The present invention provides an application of VSIG2 in the preparation of a product for treating autoimmune diseases, belonging to the field of biomedical technology. The new molecule hVSIG2 protein of the present invention significantly inhibits the activation and proliferation of T cells; animal experiments have shown that hVSIG2 protein has significant therapeutic effects on multiple sclerosis (MS) and inflammatory bowel disease (IBD), indicating that hVSIG2 protein has the effect of inhibiting immune response and improving multiple sclerosis and inflammatory bowel disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of VSIG2 in the preparation of products for treating autoimmune diseases. Background Art

[0002] Autoimmune diseases refer to a class of diseases in which the immune system attacks the body itself, causing local or systemic inflammation or tissue destruction, including multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), etc. The pathogenesis of autoimmune diseases is very complex and there is no consensus yet, but abnormal activation of autoreactive T cells is one of the important mechanisms for the occurrence of autoimmune diseases. Under normal circumstances, autoreactive T cells are suppressed to prevent attacks on self-tissues. However, in autoimmune diseases, these T cells are abnormally activated, resulting in abnormal enhancement of the immune response, thereby triggering inflammation and tissue damage. The dysfunction of T cells in autoimmune diseases may involve multiple aspects, including the activation and proliferation of T cells, and the inflammatory response caused by the attack on self-tissues.

[0003] At present, common methods for treating autoimmune diseases include drug therapy, immunomodulatory therapy and cell therapy. Drug therapy needs to be used for a long time to maintain its efficacy, but long-term use may increase the risk of side effects and adverse reactions of the drug; for example, immunosuppressants may increase the risk of infection, and non-steroidal anti-inflammatory drugs may cause gastrointestinal problems. Clinically, immunomodulatory therapy is often used to treat autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease, but the price of some immunomodulatory treatments is high, which imposes a heavy financial burden on patients; at the same time, this method is not suitable for all patients, and some patients may be insensitive or ineffective to these treatments. Clinically, cell therapy is often used to treat autoimmune diseases such as severe systemic lupus erythematosus and multiple sclerosis. This method is an invasive treatment that will increase the patient's pain and the risk of complications. Cell therapy also requires highly professional technical and equipment support, and has high requirements for medical institutions and medical staff. At present, the safety and effectiveness of cell therapy in the treatment of autoimmune diseases still need further research and verification.

[0004] VSIG2 (V-set and immunoglobulin domain containing 2) is a membrane-bound protein that belongs to the Ig superfamily. Its structure contains an IgV-type domain and an IgC-type domain, and is connected to the cytoplasm through a single transmembrane structure. In recent years, multi-omics approaches have been used to demonstrate that VSIG2 is closely associated with the progression of various human diseases. Abnormal expression of VSIG2 has been detected in intestinal biopsies of patients with irritable bowel syndrome (IBS-D), plasma of patients with acute tubular injury and interstitial fibrosis / tubular atrophy, and plasma of patients with heart failure, demonstrating that VSIG2 has the potential to serve as a biomarker for the diagnosis of many diseases. In addition, single nucleotide polymorphisms (SNPs) of VSIG2 are also correlated with the serological profile and cytokine phenotype of systemic lupus erythematosus (SLE). However, there are no reports on VSIG2 improving autoimmune diseases. Summary of the invention

[0005] The present invention provides the use of VSIG2 in the preparation of products for treating autoimmune diseases. The hVSIG2 protein significantly inhibits the activation and proliferation of T cells and has a significant improvement effect on autoimmune diseases such as multiple sclerosis and inflammatory bowel disease.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides application of VSIG2 in preparing products for treating autoimmune diseases.

[0008] Preferably, the nucleotide sequence of VSIG2 is shown as SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of VSIG2 is shown as SEQ ID NO.2.

[0010] Preferably, the autoimmune diseases include multiple sclerosis and inflammatory bowel disease.

[0011] Preferably, the VSIG2 can significantly inhibit the proliferation and activation of T cells.

[0012] Preferably, the VSIG2 can significantly inhibit the expression of inflammatory factors INF-γ, TNF-α, and IL-17a secreted by CD4+T cells, and promote the expression of IL-4.

[0013] Preferably, the dosage of VSIG2 is 0.25-15 μg / g.

[0014] The present invention provides a medicine for treating autoimmune diseases, wherein the medicine comprises the VSIG2.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable carrier of VSIG2.

[0016] Preferably, the pharmaceutical dosage forms include tablets, injections, inhalants, granules, pills, capsules, and ointments.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discovered the VSIG2 molecule for the first time, and verified from the structure and function that the VSIG2 molecule has the function of regulating T cell function, and proposed a new treatment plan for autoimmune diseases, which is an innovative treatment method. The VSIG2 protein is mainly expressed on APC cells. It binds to the corresponding receptors on T cells, inhibits T cell proliferation and activation, puts T cells in an inhibitory state, and ultimately plays a role in inhibiting immune response. The present invention prepares hVSIG2-Ig fusion protein through a set preparation method; cytological verification shows that hVSIG2-Ig fusion protein has the function of negatively regulating T cell response; and animal verification shows that hVSIG2-Ig fusion protein has a significant improvement effect on autoimmune diseases such as multiple sclerosis and inflammatory bowel disease.

[0019] The VSIG2 treatment scheme for autoimmune diseases proposed by the present invention is scalable and applicable, and can be applied to the treatment of various autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, type Ⅰ diabetes, psoriasis, autoimmune thyroiditis, etc. At the same time, the technical scheme of the present invention can also be combined with other treatment methods, such as immunosuppressants, biological agents, etc., to achieve better treatment effects.

[0020] The present invention proposes to use VSIG2 / VSIG2-R as a new immune regulation target. This technical solution has great potential in the application of autoimmune disease treatment. The development of a treatment method based on VSIG2 molecules can meet the treatment needs of patients with autoimmune diseases, improve the treatment effect and quality of life, and save medical resources and costs. At the same time, the innovation and sustainability of this technical solution also bring commercial competitiveness and market advantages to related enterprises and institutions, and have huge expected benefits and commercial value. Therefore, the technical solution of the present invention has great commercial potential and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Schematic diagram of the comparison of homology and similarity between VSIG2 and B7 family-related molecules; A: multiple sequence alignment of the extracellular domain of mVSIG2 with some known B7 and BTN family members; B: protein sequence comparison of the extracellular domains of mVSIG2 and hVSIG2, with the predicted IgV-like domain, IgC-like domain and transmembrane (TM) domain of VSIG2 marked; C: developmental tree of multiple sequence alignment of the extracellular domain of mVSIG2 with some known B7 and BTN family members; D: similarity and homology statistics of multiple sequence alignment of the extracellular domain of mVSIG2 with some known B7 and BTN family members.

[0022] Figure 2 Expression analysis diagram of VSIG2 protein; AG: expression analysis diagram of mVSIG2 protein in resting and activated immune cells; H: statistical results of expression of mVSIG2 protein in resting and activated immune cells.

[0023] Figure 3 In vitro detection of the effect of the purchased hVSIG2 protein on the function of T cells; AD: Typical flow cytometry diagrams of the co-expression of hVSIG2 and hPD-L1 in B cells, DC cells, macrophages and monocytes; EG: The effect of hVSIG2 protein on the proliferation of mouse T cells in vitro, typical diagram (E) and statistical results of ki67 expression in CD4+T cells (F) and CD8+T cells (G); HJ: The effect of hVSIG2 protein on the activation of mouse T cells in vitro, typical diagram (H) and CD4+T cells (I ) and CD69 expression statistical results of CD8+ T cells (J); KL: flow cytometry analysis of CD44loCD62Lhi and CD44hiCD62Llo expression of T cells; MP: statistical analysis of the percentages of CD44loCD62Lhi naive and CD44hiCD62Llo effector memory CD4 and CD8 T cells, initial CD4+ T cells (M), effector CD4+ T cells (N), initial CD8+ T cells (O) and effector CD8+ T cells (P).

[0024] Figure 4 The results of gel Coomassie brilliant blue and Western Blot verification of the purified hVSIG2-Ig fusion protein; 1: MW markers; 2: Coomassie blue stained hVSIG2-Ig SDS-PAGE; 3: anti-IgG2a antibody Western blot; 4: anti-hVSIG2 antibody Western blot.

[0025] Figure 5In vitro detection of the functional effect of hVSIG2-Ig fusion protein on T cells; AC: effect of hVSIG2-Ig on in vitro mouse T cell activation; DI: effect of hVSIG2-Ig fusion protein on in vitro mouse T cell proliferation: T cells were analyzed by flow cytometry for ki67 and CFSE expression after 72 hours; JK: typical flow cytometry graph of flow cytometry analysis of CD44loCD62Lhi and CD44hiCD62Llo expression of T cells; LO: statistical analysis of the percentages of CD44loCD62Lhi naive and CD44hiCD62Llo effector memory CD4 and CD8 T cells.

[0026] Figure 6 Expression analysis of putative VSIG2 receptors; AG: representative flow cytometry profiles; H: statistical analysis of VSIG2 receptor expression levels on immune cells; I: expression of CD28, CTLA-4, PD-1, BTLA, VISTA and ICOS genes; J: flow cytometry detection of the binding of biotinylated hVSIG2-Ig or control Ig to transfected cells.

[0027] Figure 7 Schematic diagram of the effect of hVSIG2-Ig on EAE mice; A: Average clinical score of EAE model mice; B: H&E staining of spinal cord tissues of two groups of mice and the statistical results of spinal cord pathology scores. C: LFB staining of spinal cord tissues of two groups of mice and the statistical results of spinal cord pathology scores; D: Immunofluorescence staining of spinal cord tissues of two groups of mice (GFAP: green, Iba1: red); E: Immunofluorescence staining of spinal cord tissues of two groups of mice (CD4, CD8: red); FG: Analysis and statistics of GFAP and Iba1 immunofluorescence staining results; HI: Analysis of CD4 and CD8 immunofluorescence staining results; JK: Typical graph of the percentage of T cells in the spleen of two groups of mice and the statistical results of CD4 and CD8; LM: Typical graph of the percentage of CD69 T cells in the spleen of two groups of mice and C Statistical results of D4+CD69+ and CD8+CD69+ activated T cells; NO: analysis of spleen cells to detect CD44loCD62Lhi naive cells, CD44hiCD62Llo effector memory cells and CD44hiCD62Lhi central memory CD4 and CD8 T cells and their statistical results; PQ: typical flow cytometry graphs of macrophages M1 and M2 polarization and statistical results; RS: percentage of CD4+CD25+FoxP3+Tregs and its statistical results; T: Kaplan-Meier curve of survival rate of the two groups of mice.

[0028] Figure 8Effect of hVSIG2-Ig on the expression of inflammatory factors in immune cells of EAE model mice; A: Expression difference of TNFα in CD4+T cells; B: Expression difference of INF-γ in CD4+T cells; C: Expression difference of IL-17A in CD4+T cells; D: Expression difference of IL-4 in CD4+T cells; EH: Flow cytometric analysis of the percentage of cytokines TNFα, INF-γ, IL-4, and IL-17A produced in CD4+T cells in spleen cells stimulated with different doses of MOG35-55.

[0029] Fig. 9 Schematic diagram of the effect of hVSIG2-Ig on IBD model mice. A: Percentage change in body weight of IBD model mice; B: DIA score of IBD model mice; CD: Colon length of two groups of mice and its statistical results; EF: H&E staining of colon tissue of two groups of mice and its statistical results; GH: Immunofluorescence staining of colon tissue of two groups of mice (Occluding: red, CD3, CD4: green); IJ: Q-PCR detection of TNF-αmRNA and IL-1βmRNA in the colon of two groups of mice; KL: Typical graph of CD69 percentage of T cells in spleen of two groups of mice and CD4 +CD69+ and CD8+CD69+ activated T cells statistical results; MN: Detection of KI67 of CD4+T cells and CD8+T cells in the spleens of the two groups of mice and the statistical results; OP: Detection of CFSE of CD4+T cells and CD8+T cells in the spleens of the two groups of mice and the statistical results; QX: Typical graphs of the percentages of TNFα, INF-γ, IL-4, and IL-17A in CD4+T cells and the statistical results; YZ: The percentage of CD4+CD25+FoxP3+Tregs and its statistical results. DETAILED DESCRIPTION

[0030] The present invention provides the use of VSIG2 in the preparation of a product for treating autoimmune diseases. The VSIG2 of the present invention can be purchased through commercial channels or obtained through the preparation method described in the embodiments of the present invention.

[0031] In the present invention, the VSIG2 is an hVSIG2-Ig fusion protein, and the specific amino acid sequence is VEVKVPTEPLSTPLGKTAELTCTYSTSVGDSFALEWSFVQPGKPISESHPILYFTNGHLYPTGSKSKRVSLLQNPPTVGVATLKLTDVHPSDTGTYLCQVNNPPDFYTNGLGLINLTVLVPPSNPLCSQSGQTSVGGSTALRCSSSEGAPKPVYNWVRLGTFPTPSPGSMVQDEVSGQLILTNLSLTSSGTYRCVATNQMGSASCELTLSVTEPSQGRVA (SEQ ID NO.2). The specific nucleotide sequence of the hVSIG2-Ig fusion protein of the present invention is: GTGGAGGTGAAGGTACCCACAGAGCCGCTGAGC ACGCCCCTGGGGAAGACAGCCGAGCTGACCTGCACCTACAGCACGTCGGTGGGAGACAGCTTCGCCCTGGAGTGGAGCTTTGTGCAGCCTGGGAAACCCATCTCTGAGTCCCATCCAATCCTGTACTTCACCAATGGCCATCTGTATCCAACTGGTTCTAAGTCAAAGCGGGTCAGCCTGCTTCAGAACCCCCCCACAGTGGGGGTGGCCACACTGAAACTGACTGACGTCCACCCCTCAGATACTGGAACCTACCTCTGCCAAGTCAACAACCCACCAGATTTCTACACCAATGGGTTGGGGCTAATCAACCTTACTGTGCTGGTTCCCCCCAGTAATCCCTTATGCAGTCAGAGTGGACAAACCTCTGTGGGAGGCTCTACTGCACTGAGATGCAGCTCTTCCGAGGGGGCTCCTAAGCCAGTGTACAACTGGGTGCGTCTTGGAACTTTTCCTACACCTTCTCCTGGCAGCATGGTTCAAGATGAGGTGTCTGGCCAGCTCATTCTCACCAACCTCTCCCTGACCTCCTCGGGCACCTACCGCTGTGTGGCCACCAACCAGATGGGCAGTGCATCCTGTGAGCTGACCCTCTCTGTGACCGAACCCTCCCAAGGCCGAGTGGCC (shown in SEQ ID NO.1).In the present invention, a tag protein (FC tag protein) needs to be introduced during the purification of the hVSIG2-Ig fusion protein, so the hVSIG2-Ig fusion protein contains a FC tag protein. The nucleotide sequence of the hVSIG2-Ig fusion protein of the present invention is the hVSIG2 extracellular region sequence. The hVSIG2 extracellular region sequence of the present invention is obtained by PCR amplification using the full-length cDNA of the hVSIG2 extracellular region as a template and the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4. The amino acid sequence of the hVSIG2-Ig fusion protein of the present invention is the 24th to 243rd amino acids of the full amino acid sequence of hVSIG2. The nucleotide sequences of the primer pairs of the present invention are: F 5'-3': GTCA CGAATTCGGCGATCGCGTGGAGGTGAAGGTACCCAC (SEQ ID NO.3), R 5'-3': GCGGCCGCGTACGCGTTCGGCCTTGGGAGGGTTCGG (SEQ ID NO.4).

[0032] In the present invention, the autoimmune diseases include multiple sclerosis and inflammatory bowel disease. The VSIG2 described in the present invention can significantly inhibit the proliferation and activation of T cells, inhibit the secretion of inflammatory factors INF-γ, TNF-α, IL-17a by CD4+T cells, and promote the expression of IL-4, thereby achieving the purpose of treating multiple sclerosis and inflammatory bowel disease autoimmune diseases. The dosage of VSIG2 described in the present invention is 0.25 to 15 μg / g, preferably 1 to 10 μg / g. In the mouse experiment of the present invention, the dosage of VSIG2 is preferably 1.25 μg / g.

[0033] The present invention explores that VSIG2 is upregulated in activated APCs, binds to corresponding receptors on T cells, inhibits T cell proliferation and activation, and inactivates T cells, ultimately playing a role in inhibiting the reaction. The hVSIG2-Ig fusion protein prepared by the present invention can inhibit the activation and proliferation of T cells, thereby inhibiting over-activated autoimmune reactions, and reducing inflammatory reactions and tissue damage.

[0034] The present invention injects hVSIG2-Ig fusion protein into autoimmune disease mouse models (EAE mouse model and IBD mouse model), and evaluates the effectiveness and specificity of hVSIG2-Ig fusion protein in treating multiple sclerosis by detecting the neurological function, inflammatory factor levels, histopathological changes, T cell activity and proliferation in the spleen of the EAE mouse model. The effectiveness and specificity of hVSIG2-Ig fusion protein in treating inflammatory bowel disease are evaluated by detecting the weight changes, intestinal function, inflammatory factor levels, histopathological changes, T cell activity and proliferation in the spleen of the IBD mouse model. The present invention has been verified as above and shows that the hVSIG2-Ig fusion protein has a significant improvement effect on the neurological function of multiple sclerosis and the intestinal function of inflammatory bowel disease.

[0035] The present invention also provides a drug for treating autoimmune diseases, the drug comprising the VSIG2. The drug of the present invention also comprises a pharmaceutically acceptable carrier of the VSIG2. The drug dosage forms of the present invention include but are not limited to tablets, injections, inhalants, granules, pills, capsules, and liniments.

[0036] In the present invention, unless otherwise specified, all components are commercially available products well known to those skilled in the art.

[0037] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Comparison of homology and similarity between VSIG2 and B7 family-related molecules

[0040] The amino acid sequences of VSIG2, B7 family and BTN family members were downloaded from the Uniprot official website, and the sequences were aligned using Multiple Sequence Alignment by CLUSTALW (Multiple Sequence Alignment-CLUSTALW (genome.jp)), and then analyzed and statistically analyzed. At the same time, MEGA11 was used to construct a developmental tree. The results of comparative analysis and developmental tree construction are shown in Figure 1 .

[0041] Depend on Figure 1 The results showed that the extracellular domain of mVSIG2 had high similarity and homology with some known B7 and BTN family members in multiple sequence alignment ( Figure 1 A); the sequence homology and similarity of mVSIG2 and hVSIG2 are 81% and 94%, respectively ( Figure 1 B); The extracellular domain of mVSIG2 is close to B7-1 and B7-2 ( Figure 1 C). The extracellular domain of mVSIG2 has 19%, 19%, 18%, 23%, 16%, 21%, 15%, 18%, 17% and 16% homology and 68%, 67%, 67%, 69%, 67%, 69%, 66%, 67%, 67% and 65% similarity with the extracellular domains of mouse B7-1, B7-2, B1-H2, B7-H3, B7-H4, BTN1A1, VSIG2, ICOSL, PD-L1 and PD-L2, respectively ( Figure 1 D). It can be seen that VSIG2 has a high degree of similarity and homology with B7 and BTN family members, and it may be a B7 family-related molecule.

[0042] 2. Expression pattern of VSIG2 protein in immune cells

[0043] To obtain activated B cells, DCs, monocytes and macrophages, spleen immune cells were cultured with LPS (5 μg / ml) for 3 days. To obtain activated T cells, cells were incubated with anti-CD3 antibody (1 μg / ml) for 3 days. Resting and activated immune cells were stained with anti-VSIG2Ab or isotype antibody and anti-CD4, CD8, CD19, B220, CD11c, F4 / 80 and CD11b antibodies, and flow cytometry was used to identify the expression level of VSIG2 on different immune cells.

[0044] The specific detection steps are as follows:

[0045] Preparation of spleen immune cell suspension: (1) C57BL / 6 mice (purchased from the Animal Experiment Center of Guizhou Medical University, production license number: SCXK(Gui)2023-0002) were killed by cervical dislocation. After disinfection, the spleen was removed and soaked in a clean PBS solution. (2) The spleen was placed on a glass slide and gently ground until there were no obvious red lumps. (3) The cell suspension was collected in a 15 mL centrifuge tube, centrifuged at 300 g for 5 min, and the supernatant was discarded. (4) 4 mL of 1× red blood cell lysis buffer was added to resuspend the cells. After lysis at room temperature for 2 to 3 minutes, 10 mL of PBS was immediately added, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. (5) The spleen cells were resuspended in cell staining buffer, the cell suspension was filtered again with a 200-mesh sieve, counted, and the cell concentration was adjusted to 1×10 7 / mL.

[0046] Flow cytometry: (1) Take out the 96-well plate pre-coated with LPS (5 μg / ml) and CD3 antibody (1 μg / ml) from the 4°C refrigerator, and add the prepared single cell suspension of normal C57BL / 6 mouse spleen into the marked wells at 200 μl / well. Set up 3 independent sample wells for each group of culture conditions to repeat the experiment. Incubate in a 37°C, 5% CO2 cell culture incubator for 3 days. On the third day, prepare the immune cell suspension again to obtain resting cells. (2) Take out the 96-well plate from the incubator and centrifuge at 3000 rpm for 5 min to remove the supernatant. (3) Add monoclonal direct-labeled primary antibodies APC-CD4, PerCP / Cy5.5-CD8a, PE-CD11b, PE-CD11c, PE-CD19, PerCP / Cy5.5-B220, PerCP / Cy5.5-F4 / 80, FITC-VSIG2 and isotype control to each well at a dilution ratio of 1:100. Incubate at 4℃ in the dark for 45 minutes. (4) Add 200μl 1×PBS to each well to resuspend the cells, centrifuge and remove the supernatant; add 100μl 1×PBS to each well to resuspend the cells, transfer to a flow tube and analyze using a flow cytometer.

[0047] like Figure 2 As shown in the typical figure, the results show that VSIG2 is expressed in resting immune cells, and its expression level is significantly increased in activated T cells and antigen presenting cells (APCs). Figure 2 A- Figure 2 G). Statistical analysis results showed that Figure 1 To Figure 2 H). This indicates that VSIG2 is expressed in resting immune cells and is more prominently expressed in activated T cells and antigen-presenting cells (APCs).

[0048] 3. In vitro detection of the effect of the purchased hVSIG2 protein (sino biological, Cat: 11543-H08H) on the function of T cells: hPD-L1 was used as a positive control to determine the effect of hVSIG2 protein on the function of T cells.

[0049] The steps for detecting the co-expression of hVSIG2 and hPD-L1 (purchased from sino biological, Cat: 10084-H08H-B) in immune cells are as follows: (1) Prepare mouse spleen single cell suspension as described above, take out the 96-well plate pre-coated with LPS (5μg / ml) from the 4°C refrigerator, pour out the coated liquid, and add the prepared normal C57BL / 6 mouse spleen single cell suspension to the marked wells at 200μl / well in turn. Set up 3 independent sample wells for each group of culture conditions for experimental technical repetition. Incubate in a cell culture incubator at 37°C, 5% CO2 for 3 days. (2) Take out the 96-well plate from the incubator and centrifuge to remove the supernatant. (3) Monoclonal direct-labeled primary antibodies PerCP / Cy5.5-220, PE-CD11b, PE-CD11c, PerCP / Cy5.5-F4 / 80, FITC-VSIG2 and APC-PD-L1 were added to each well at a dilution ratio of 1:100. Incubate at 4°C in the dark for 45 min; (4) Add 200 μl 1× PBS to each well to resuspend the cells, centrifuge and remove the supernatant, add 100 μl 1× PBS to each well to resuspend the cells, transfer to a flow tube and analyze using a flow cytometer.

[0050] Flow cytometry detection of the effect of hVSIG2 protein on effector T cell differentiation: (1) Anti-CD3 and hVSIG2 protein or Control Ig protein (purchased from sinobiological, Cat: 51094-MNAH) were coated in 96-well plates according to the corresponding equimolar mass Fc protein. (2) The spleen single cell suspension of normal C57BL / 6 mice was plated into a 96-well plate, cultured in an incubator for 24 hours, and the supernatant was discarded after centrifugation. (3) A 1:100 dilution ratio of monoclonal direct labeled primary antibodies APC-CD4, FITC-CD8a, and PE-CD69 were added to each well of cells to detect spleen T cell activation; PE-CD44 and FITC-CD62L staining were used to detect the ratio of spleen effector T cells and naive T cells; incubated for 45 minutes, washed once with PBS, and resuspended for flow cytometry. (4) Flow cytometry results Statistical analysis of the percentages of CD44loCD62Lhi naive and CD44hiCD62Llo effector memory CD4 and CD8 T cells, and the percentage of initial CD4+ T cells ( Figure 3 M), percentage of effector CD4+ T cells ( Figure 3 N), initial CD8+T cell percentage ( Figure 3 O) and percentage of effector CD8+ T cells ( Figure 3 P).

[0051] like Figure 3As shown, VSIG2 and PD-L1 are co-expressed in activated antigen-presenting cells APCs, with a co-expression percentage of 12.4±0.45 in B cells. The co-expression percentage in DC cells is 32±0.52. The co-expression percentage in macrophages is 74.8±0.62. The co-expression percentage in monocytes is 58.6±0.67. Mouse T cells were intervened by hVSIG2 or hPD-L1 at different concentrations (1600ng / ml, 3200ng / ml, 6400ng / ml). After 24 hours of culture, the expression changes of CD69, a molecule related to T cell activation, were detected by FACS. The results showed that different concentrations of hVSIG2 and hPD-L1 could significantly inhibit the activation of T cells. After 72 hours of cell culture, the proliferation of T cells (Ki67) was detected by FACS. The results showed that different concentrations of hVSIG2 or hPD-L1 could significantly inhibit the proliferation of T cells. FACS detected the expression of CD62L, a marker of initial T cells, and CD44, a marker of effector T cells. The results showed that different concentrations of hVSIG2 and hPD-L1 could significantly inhibit the differentiation of T cells into effector T cells, indicating that hVSIG2 has the function of negatively regulating T cell responses.

[0052] Example 2 Preparation of hVSIG2-Fc fusion protein

[0053] (1) Obtaining the VSIG2 gene sequence: The full-length gene sequence of the hVSIG2 extracellular region was retrieved from NCBI (NM_014312.4). The hVSIG2 full-length cDNA (purchased from Youbao Biotechnology, product number: G109787) was used as a template and the hVSIG2 extracellular region sequence was amplified by PCR using the primer pair F 5'-3': GTCACGAATTCGGCGATCGCGTGGAGGTGAAGGTACCCAC (SEQ ID NO. 3) and R 5'-3': GCGGCCGCGTACGCGTTCGGCCTTGGGAGGGTTCGG (SEQ ID NO. 4). The hVSIG2 target gene (SEQ ID NO. 1) was recovered by gel excision.

[0054] (2) Construction of hVSIG2 gene expression plasmid: The pCMV6-AC-FC-S expression vector was cut into linear fragments using MIU1 and Sigf1 enzymes, and the hVSIG2 target gene was then ligated to the pCMV6-AC-FC-S plasmid using In-Fusion HD Enzyme Premix DNA ligase.

[0055] (3) Transfection: The constructed hVSIG2 plasmid was transfected into HEK-293F cells, and G418 geneticin at a concentration of 200 μg / ml was used to select cell lines with stable expression.

[0056] (4) Preparation and verification of hVSIG2-Ig fusion protein: After culturing the stably expressed cell line with FreeStyleTM293 expression medium for 2 weeks, the supernatant was collected and concentrated, and the protein was purified by ProteinA / G protein purification column. hVSIG2 was attached to the purification column through the Fc tag, and the hVSIG2-Ig fusion protein was obtained by eluting the column. The results were as follows: Figure 4 The results showed that a single band of protein that could bind to anti-FCAb and anti-hVSIG2Ab was obtained, which was the purified hVSIG2-Ig fusion protein (hVSIG2-FC fusion protein). After sequencing, the nucleotide sequence of the hVSIG2-Fc fusion protein was shown in SEQ ID NO.1.

[0057] Example 3

[0058] 1. In vitro detection of the effect of the prepared hVSIG2-Ig fusion protein on T cell function

[0059] Flow cytometry to detect the effect of hVSIG2-Ig fusion protein on T cell differentiation: In order to verify whether the prepared hVSIG2-Ig fusion protein has function on T cell response, mouse T cells were intervened by hVSIG2-Ig fusion protein and Control Ig (FC-tagged protein, purchased from Sino biological, Cat: 51094-MNAH) at different concentrations (1600ng / ml, 3200ng / ml, 6400ng / ml). After 24 hours of culture, the expression changes of CD69, a molecule related to T cell activation, were detected by FACS. The steps are as follows:

[0060] (1) Anti-CD3 and hVSIG2-Ig proteins or Control Ig proteins were coated in 96-well plates according to the corresponding equimolar mass of Fc proteins.

[0061] (2) The spleen single cell suspension of normal C57BL / 6 mice was plated into a 96-well plate and cultured in an incubator for 24 hours. The supernatant was then discarded after centrifugation.

[0062] (3) Antibodies APC-CD4, FITC-CD8a, and PE-CD69 were added to each well to detect splenic T cell activation; PE-CD44 and FITC-CD62L staining were used to detect the ratio of splenic effector T cells and naive T cells; the cells were incubated for 45 min, washed once with PBS, and resuspended for flow cytometry.

[0063] (4) Flow cytometry results Statistical analysis of the percentages of CD44loCD62Lhi naive and CD44hiCD62Llo effector memory CD4 and CD8 T cells, and the percentage of initial CD4+ T cells ( Figure 5 L), percentage of effector CD4+ T cells ( Figure 5 M), initial CD8+T cell percentage ( Figure 5 N) and percentage of effector CD8+ T cells ( Figure 5 O).

[0064] The effect of hVSIG2-Ig protein on T cell proliferation was detected by flow cytometry. The steps are as follows:

[0065] (1) Anti-CD3 and hVSIG2-Ig proteins or Control Ig proteins were coated in 96-well plates according to the corresponding equimolar mass Fc proteins. The Ki67 group was directly plated with the spleen single cell suspension of normal C57BL / 6 mice. The CFSE group was incubated with CFSE for 15 minutes for staining and then plated into 96-well plates. After 5 days of incubation in an incubator, the cells were collected and the supernatant was removed by centrifugation.

[0066] (2) Add antibodies APC-CD4 and FITC-CD8a to each well of cells and incubate for 45 minutes.

[0067] (3) After resuspension, the CFSE group was analyzed on a flow cytometer for the proliferation ratio of CD4+T cells and CD8+T cells. In the Ki67 group, 4% paraformaldehyde was added to each well for fixation for 10 min, and then 100 μl of 0.2% TrionX-100 was punched on ice for 10 min. PE-Ki67 antibody was added to each well, and the cells were incubated on ice for 45 min. The cells were then tested on a flow cytometer for cell proliferation.

[0068] The results are as follows Figure 5As shown, different concentrations of hVSIG2-Ig fusion protein can significantly inhibit the activation of T cells. After 72 hours of cell culture, the proliferation of T cells (CFSE, Ki67) was detected by FACS, and the results showed that different concentrations of hVSIG2 can significantly inhibit the proliferation of T cells. FACS detected the expression of the initial T cell marker CD62L and the effector T cell marker CD44 of T cells, and the results showed that different concentrations of hVSIG2-Ig fusion protein can significantly inhibit the differentiation of T cells into effector T cells. This shows that the hVSIG2-Ig fusion protein prepared by the present invention has the function of negatively regulating T cell response.

[0069] 2. Expression of putative VSIG2 receptors in immune cells

[0070] The expression of putative VSIG2 receptors in immune cells was investigated by flow cytometry. The specific steps are as follows:

[0071] The spleen immune cell suspension of C57BL / 6 mice was obtained by the same steps as described in step 2 of Example 1.

[0072] Flow cytometry detection: 1) Take out the 96-well plate pre-coated with LPS (5μg / ml) and CD3 antibody (1μg / ml) from the 4℃ refrigerator, pour out the coating liquid, and add the prepared single cell suspension of normal C57BL / 6 mouse spleen to the marked wells at 200μl / well. Set up 3 independent sample wells for each group of culture conditions to repeat the experimental technology. Incubate in a cell culture incubator at 37℃, 5% CO2 for 3 days. On the third day, prepare the immune cell suspension again to obtain the resting cells. 2) Take out the 96-well plate and centrifuge to remove the supernatant. 3) Add 50ul CD16 / 32 (dilution ratio 1:100) to each well to block non-specific Ig on the cell surface. After incubation for 10 minutes, add 200μl 1×PBS to each well to resuspend the cells and centrifuge to remove the supernatant. 4) Add 100ul of pre-biotinylated hVSIG2-Ig fusion protein (biotin is covalently linked to hVSIG2-Ig fusion protein) and Control Ig protein (FC-tagged protein, purchased from sino biological, Cat: 51094-MNAH) and incubate on ice for 1h; add 200μl 1×PBS to each well to resuspend the cells and centrifuge to remove the supernatant. 5) Resuspend the cell pellet obtained in each tube with 100μl 0.5% BSA, and add monoclonal direct-labeled primary antibodies APC-CD4, PerCP / Cy5.5-CD8a, APC-CD11b, FITC-CD11c, APC-CD19, PerCP / Cy5.5-B220, FITC-F4 / 80 and PE-labeled streptavidin to each tube at a dilution ratio of 1:100; incubate at 4℃ in the dark for 45min. 6) Add 200 μl 1×PBS to each well to resuspend the cells, centrifuge to remove the supernatant, add PBS to each well to resuspend the cells, transfer to a flow tube and detect using a flow cytometer.

[0073] Flow cytometry was used to detect the binding of biotinylated hVSIG2-Ig or Control Ig to transfected cells. The specific steps are as follows:

[0074] (1) The purchased CD28 (sino biological, Cat: HG11524-ACG), CTLA-4 (sinobiological, Cat: HG11159-ACG), PD-1 (sino biological, Cat: HG10377-CH), BTLA (sinobiological, Cat: HG11895-UT), VISTA (sino biological, Cat: HG13482-ACG) and ICOS (sinobiological, Cat: HG10344-ACR) gene plasmids were transfected into empty HEK-293F cells. (2) According to the resistance gene screening, the transfected gene-positive HEK-293F cells were obtained, and the corresponding CD28, CTLA-4, PD-1, BTLA, VISTA and ICOS protein antibodies were used for staining detection, and a blank control was set up. The positive rate of transfected genes was detected by flow cytometry. (3) After obtaining the purified transfected gene-positive HEK-293F cells, biotinylated hVSIG2-Ig or control Ig was bound to them, stained with PE-labeled streptavidin, and flow cytometry was used to detect the binding of hVSIG2-Ig to the transfected cells.

[0075] like Figure 6 As shown: The expression level of the putative VSIG2 receptor in activated T cells and antigen presenting cells (APCs) was significantly increased ( Figure 6 A- Figure 6 H). The expression of CD28, CTLA-4, PD-1, BTLA, VISTA and ICOS proteins in 293F cells was significantly upregulated, indicating that the plasmid transfection was successful ( Figure 6 I). hVSIG2-Ig does not bind to CD28, CTLA-4, PD-1, BTLA, VISTA and ICOS ( Figure 6 J). It can be seen that the putative VSIG2 receptor is significantly upregulated on activated T cells and does not bind to receptors such as CD28, PD-1, CTLA-4, BTLA, VISTA and ICOS, suggesting that VSIG2 binds to the VSIG2 receptor and may be a new immune checkpoint molecule.

[0076] Example 4 Effect of hVSIG2-Ig fusion protein on the autoimmune disease EAE

[0077] Construction of EAE mouse model, neurological function score and survival rate statistics: C57BL / 6 mice were immunized with 200μg MOG35-55 emulsified in CFA and 500ng purified Bordetella pertussis toxin. The constructed model mice were divided into a treatment group (hVSIG2-Ig fusion protein) and a control group (Control Ig protein, source as above). From day 13, the model mice were intraperitoneally injected with 25μg hVSIG2-Ig or Ig protein three times a week for a total of 5 weeks. The development of EAE was monitored for 42 days, and the neurological function and survival rate of the mice were recorded every day.

[0078] HE staining and LFB staining of mouse spinal cord tissue: spinal cord tissues of hVSIG2-Ig and Control Ig mice were obtained, paraffin-embedded and sectioned, dewaxed, HE stained with hematoxylin for nuclei and eosin for cytoplasm, dehydrated and sealed for microscopic observation. LFB staining was stained with Luxol FastBlue staining solution, separated by Luxol differentiation solution, counterstained with tar violet staining solution, dehydrated and sealed for microscopic observation.

[0079] Immunofluorescence staining of GFAP, IBA1, CD4, and CD8 in mouse spinal cord tissue: Spinal cord tissues of hVSIG2-Ig and Control Ig mice were obtained, embedded in wax, and sliced. The slices were dewaxed and repaired with antigen repair solution. After blocking with goat serum, the primary antibodies GFAP, IBA1, CD4, and CD8 (1:1000) were incubated at 4°C overnight. The next day, the slides were washed with PBS and the secondary antibodies goat anti-rabbit 488 and cy3 were added respectively. The slides were incubated at room temperature in the dark for 2 hours. After washing with PBS, the nuclei were stained with DAPI for 10 minutes. After sealing, the slides were observed under a fluorescence microscope and images were collected. Statistical results of the number of GFAP-positive cells ( Figure 7 F) Statistical results of IBA1 positive cells ( Figure 7 G) CD4 positive cell count results ( Figure 7 H) Statistical results of CD8 positive cells ( Figure 7 I).

[0080] Flow cytometry was used to detect the number and percentage of T cells in the spleens of the two groups of mice: spleens of the two groups of mice were taken and made into single-cell suspensions, which were added to 96-well plates according to the labels. APC-CD4 and FITC-CD8a were added to each well and incubated for 45 minutes. Changes in the number and percentage of T cells were detected by flow cytometry.

[0081] Flow cytometry was used to detect T cell activation in the spleens of the two groups: spleens of the two groups of mice were taken and made into single-cell suspensions, which were added to 96-well plates according to the labels. APC-CD4, FITC-CD8a, and PE-CD69 were added to each well and incubated for 45 minutes. The expression changes of CD4+T cells and CD8+T cells CD69 were detected by flow cytometry.

[0082] Flow cytometry was used to detect the differentiation of effector T cells in the spleens of the two groups: spleens of the two groups of mice were taken and made into single-cell suspensions, which were added to 96-well plates according to the labels. APC-CD4, PerCP / Cy5.5-CD8a, PE-CD44 and FITC-CD62L were added to each well and incubated for 45 minutes. The expression of initial T cell marker CD62L and effector T cell marker CD44 in CD4+T cells and CD8+T cells was detected by flow cytometry.

[0083] Flow cytometry was used to detect the polarization of macrophages in the spleens of the two groups: spleens of the two groups of mice were taken, single-cell suspensions were made, and added to 96-well plates according to the labels. PerCP / Cy5.5-F4 / 80 and FITC-MHC-II were added to each well and incubated for 45 minutes. The plates were fixed with 4% paraformaldehyde for 10 minutes, and then 100 μl of 0.2% TrionX-100 was used to punch the plates on ice for 10 minutes. AlexaFluor647-CD206 antibody was added to each well and the plates were put on a flow cytometer to detect the expression of M1 macrophage MHC-II and M2 macrophage CD206 in the macrophages.

[0084] Flow cytometry was used to detect the expression of Tregs in the spleens of the two groups: spleens of the two groups of mice were taken and made into single cell suspensions, which were added into 96-well plates according to the labels. APC-CD4 and PE-CD25 were added into each well and incubated for 45 min. The plates were fixed with 4% paraformaldehyde for 10 min, and then 100 μl of 0.2% TrionX-100 was used to punch the plates on ice for 10 min. FITC-FOXP3 antibody was added into each well and the plates were put on flow cytometry machine to detect the expression of Tregs in the immune cells of the spleens of the two groups.

[0085] Flow cytometry was used to detect the expression of inflammatory factors in the spleens of the two groups: spleens of the two groups of mice were taken, single-cell suspensions were made, and added to 96-well plates according to the labels. APC-CD4 was added to each well and incubated for 45 minutes, fixed with 4% paraformaldehyde for 10 minutes, and then 100 μl of 0.2% TrionX-100 was punched on ice for 10 minutes. PE-TNF-α, FITC-INF-γ, FITC-IL-17A, and PE-IL-4 antibodies were added to each well, and the expression of inflammatory factors in CD4+T cells of the two groups was detected on a flow cytometer.

[0086] Flow cytometry was used to detect the expression of inflammatory factors caused by nonspecific stimulation in the spleens of the two groups: spleens of the two groups of mice were taken and made into single cell suspensions, which were added to 96-well plates pre-coated with different doses of MOG35-55 according to the markers. After 3 days of cell culture, the 96-well plates were taken out. APC-CD4 was added to each well for incubation for 45 minutes, fixed with 4% paraformaldehyde for 10 minutes, and then 100 μl of 0.2% TrionX-100 was punched on ice for 10 minutes. PE-TNF-α, FITC-INF-γ, FITC-IL-17A, and PE-IL-4 antibodies were added to each well, and the expression of inflammatory factors in CD4+T cells of the two groups was detected by flow cytometry.

[0087] like Figure 7 and 8 As shown in the figure, clinical scores showed that after 4 weeks of intraperitoneal injection of hVSIG2-Ig fusion protein (25ug / time, 3 times / week), the clinical scores of the treatment group were significantly lower than those of the control group. The results of H&E staining and LFB staining showed that the degree of inflammatory cell aggregation in the spinal cord of the treatment group was reduced compared with the control group, the myelin sheath was relatively neatly arranged, the myelin sheath density was high, and the degree of myelin loss was significantly reduced. In addition, immunofluorescence results showed that the number of activated astrocytes and microglia in the spinal cord of mice in the treatment group was significantly reduced compared with the control group, and the number of CD4+T cells and CD8+T cells in the spinal cord was significantly reduced. Flow cytometry was used to detect immune cells in the spleen of each group. Compared with the control group, the percentage of CD4+T cells and CD8+T cells in the spleen of mice in the treatment group was significantly reduced. hVSIG2-Ig significantly inhibited the activation of CD4+T cells and CD8+T cells, as well as the differentiation of T cells into effector T cells. At the same time, hVSIG2-Ig promoted the differentiation of macrophages into M2 macrophages, while inhibiting the differentiation of M1 macrophages. At the same time, hVSIG2-Ig also promoted a significant increase in the percentage of Treg cells. The survival rates of the two groups of mice were statistically analyzed, and the survival rate of mice in the treatment group was higher than that in the control group. In addition, the expression of inflammatory-related factors in the spleen of each group was detected, and it was found that hVSIG2-Ig could significantly inhibit the secretion of TNF-α, INF-γ, and IL-17A by CD4+T cells, while promoting the production of IL-4. After stimulation with different doses of MOG35-55, the inflammatory factors INF-γ, TNF-α, and IL-17a in CD4+T cells in the VSIG2-Ig group were significantly reduced, while IL-4 in CD4+T cells was increased. This shows that hVSIG2-Ig can reduce the severity of the autoimmune disease EAE in mice by inhibiting T cell function.

[0088] Example 5 Effect of hVSIG2-Ig fusion protein on autoimmune disease IBD

[0089] Construction of IBD mouse model: C57BL / 6 mice were fed with 2.5% DSS. From day 3 onwards, mice were intraperitoneally injected with 25 μg hVSIG2-Ig (experimental group) or Ig protein (control group), once every other day, for a total of 3 injections. The development of IBD was monitored for 9 days. The weight and DIA scores of mice were recorded, and the colon length was measured on day 9.

[0090] HE staining of colon tissue: colon tissues of mice in the hVSIG2-Ig and Control Ig groups were collected, embedded in paraffin and sectioned. The sections were dewaxed and HE stained with hematoxylin to stain the nucleus and eosin to stain the cytoplasm. The sections were dehydrated and mounted for observation under a microscope.

[0091] Colon tissue Q-PCR detection: colon tissues of hVSIG2-Ig and Control Ig mice were collected, total RNA was extracted, reverse transcribed into cDNA, primers TNF-α and IL-1β were designed, and samples were loaded on the machine for PCR amplification, and the obtained data were statistically analyzed.

[0092] Occluding, CD4, and CD8 immunofluorescence staining of colon tissue: The colon tissues of the hVSIG2-Ig and Control Ig groups of mice were collected, embedded in wax, and sliced. The slices were dewaxed and repaired with antigen repair solution. After blocking with goat serum, the primary antibodies of Occluding, CD4, and CD8 (1:1000) were incubated at 4°C overnight. The next day, the slides were washed with PBS and the secondary antibodies goat anti-rabbit 488 and cy3 were added respectively. The slides were incubated at room temperature in the dark for 2 hours. After washing with PBS, the nuclei were stained with DAPI for 10 minutes. After sealing, the slides were observed under a fluorescence microscope and images were collected. Statistical results of the number of occluding positive cells ( Fig. 9 G) Statistical results of CD4 and CD8 positive cells ( Fig. 9 H).

[0093] Flow cytometry was used to detect T cell activation in the spleens of the two groups of mice: spleens of the two groups of mice were taken and made into single-cell suspensions, which were added to 96-well plates according to the labels. APC-CD4, FITC-CD8a, and PE-CD69 were added to each well and incubated for 45 minutes. The expression changes of CD4+T cells and CD8+T cells CD69 were detected by flow cytometry.

[0094] Flow cytometry was used to detect T cell proliferation in the spleens of the two groups: (1) The spleens of the two groups of mice were taken and single cell suspensions were prepared. The suspensions were added to 96-well plates according to the labels. After CFSE staining, the suspensions were cultured for 3 days before detection. Antibodies APC-CD4 and FITC-CD8a were added to each well of the KI67 group and incubated for 45 minutes. 4% paraformaldehyde was added for fixation for 10 minutes, and then 100 μl of 0.2% TrionX-100 was used to punch the wells on ice for 10 minutes. PE-Ki67 antibody was added to each well and incubated on ice for 45 minutes. The cells were placed on a flow cytometer to detect cell proliferation. After the third day, antibodies APC-CD4 and PerCP / Cy5.5-CD8a were added to each well of the cells, and FITC-CFSE was detected on the machine. Statistics of CD4+T cells and CD8+T cells KI67 results ( Fig. 9 N). CFSE statistical results of CD4+T cells and CD8+T cells ( Fig. 9 P).

[0095] Flow cytometry was used to detect the expression of inflammatory factors in the spleens of the two groups: spleens of the two groups of mice were taken and made into single cell suspensions, which were added to 96-well plates according to the labels. APC-CD4 was added to each well and incubated for 45 minutes, fixed with 4% paraformaldehyde for 10 minutes, and then 100 μl of 0.2% TrionX-100 was punched on ice for 10 minutes. PE-TNF-α, FITC-INF-γ, FITC-IL-17A, and PE-IL-4 antibodies were added to each well. The expression of inflammatory factors in CD4+T cells of the two groups was detected by flow cytometry. Statistical results of TNF-α expression level in CD4+T Fig. 9 U); Statistical results of INF-γ expression level in CD4+T cells ( Fig. 9 V); Statistical results of IL-17A expression level in CD4+T cells ( Fig. 9 W); Statistical results of IL-4 expression level in CD4+T cells ( Fig. 9 X).

[0096] like Fig. 9As shown in the results, the DIA score and weight change rate of the treatment group were significantly lower than those of the control group, and the treatment group significantly inhibited the shortening of the colon length. H&E staining results showed that the structure of colonic epithelial cells in the treatment group was more regular, the integrity of the intestinal mucosal layer was more complete, and the degree of inflammatory cell infiltration was reduced compared with the control group. In addition, immunofluorescence results showed that the expression of tight junction protein occluding in the colon of mice in the treatment group was significantly increased compared with the control group, and the number of CD3+T cells and CD4+T cells positive cells was significantly reduced. Q-PCR results showed that the expression levels of TNF-αmRNA and IL-1βmRNA in the treatment group were significantly reduced. Flow cytometry results showed that compared with the control group, hVSIG2 significantly inhibited the activation and proliferation of CD4+T cells and CD8+T cells. At the same time, it promoted a significant increase in the percentage of Treg cells. In addition, hVSIG2 can significantly inhibit the secretion of TNF-α, INF-γ, and IL-17A by CD4+T cells (P<0.01), while promoting the production of IL-4. This indicates that hVSIG2-Ig can reduce the severity of the autoimmune disease IBD in mice by inhibiting T cell activation and proliferation and the release of inflammatory factors.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of VSIG2 protein in the preparation of products for the treatment of autoimmune diseases; The autoimmune diseases are multiple sclerosis and inflammatory bowel disease; The amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the VSIG2 protein is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The VSIG2 protein can significantly inhibit the proliferation and activation of T cells.

4. The use according to claim 1, characterized in that The VSIG2 protein can significantly inhibit the expression of inflammatory factors INF-γ, TNF-α, and IL-17a secreted by CD4+T cells, and promote the expression of IL-4.

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