CN112940125B

CN112940125B - anti-VISTA protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN112940125B
Application number: CN202110449149.8A
Authority: CN
Inventors: 杨清海; 陈惠玲; 王小亚; 付利利; 陈滢; 李婷
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2022-04-12
Anticipated expiration: 2041-04-25
Also published as: CN112940125A

Abstract

The present invention relates to a monoclonal antibody capable of recognizing human VISTA antigen, a secretory cell line, its preparation method and its use in immune detection. The above technical scheme selects the 269th to 289th amino acids at the C-terminus of the VISTA protein as the antigenic peptide, and the immunogen obtained after coupling with KLH is used to immunize the mouse. Monoclonal antibody mouse hybridoma cell line 11A4, and anti-VISTA protein monoclonal antibody secreted by this cell line. The antibody obtained in this protocol has high specificity and sensitivity, can specifically recognize cells expressing VISTA protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

anti-VISTA protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-VISTA protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

VISTA (V-type immunoglobulin domain-associating promoter of T-cell activation), a recently discovered immune negative regulatory checkpoint, limits the ability of T cells to effectively attack tumors. Human VISTA is 90% homologous to mouse. Like murine, human VISTA is mostly extracted from hematopoietic tissues or tissues infiltrated with a large number of leukocytes, which is an important hint for VISTA immune-related functions. The expression of VISTA protein in humans has a clear correlation with autologous blood supply, with the highest levels of expression in placenta, lung, spleen and leukocytes, higher on the cellular level with macrophages, spleen CD11B + monocytes, CD11c + dendritic cells, CD4+ T cells and CD8+ T cells, and less expression on B cells.

In recent years, a new target molecule with definite functions in the aspect of T cell immunosuppression appears, namely an immune checkpoint molecule T cell activation inhibitor immunoglobulin variable region domain (VISTA), and previous animal experiments prove that: the VISTA target is blocked, and the combined use of VISTA and other immune checkpoint inhibitors, such as PD-1 (programmed death factor), can kill tumor. VISTA has homology with PD-L1, and VISTA has different signaling pathways with PD-1/PD-L1 to suppress T cell immune activity.

In studies of the mechanism of acquired resistance in tumor immunotherapy against PD-1 or CTLA-4, VISTA was found to probably represent another compensatory inhibitory pathway. Combination blockade with VISTA and PD-1/CTLA4 may be a promising new option for cancer treatment when acquired immune resistance is present in anti-PD-1/CTLA 4 treatment of tumors. VISTA immune checkpoint has gradually become a hotspot of tumor immunosuppressant therapy research make internal disorder or usurp, and has been reported in related studies in tumors such as lung cancer, gastric cancer, melanoma, oral squamous cell carcinoma, prostate cancer and the like.

Disclosure of Invention

The invention provides an anti-VISTA protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes VISTA protein.

Further, the monoclonal antibody is mouse IgG_2aSubtype monoclonal antibodies.

Furthermore, the anti-VISTA monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20766.

The inventor also provides a preparation method of the anti-VISTA protein monoclonal antibody, which comprises the steps of selecting an amino acid sequence (269 th to 289 th) shown by SEQ ID number 1 in VISTA protein, adding a cysteine at the carboxyl terminal of the amino acid sequence, and coupling the amino acid sequence with a carrier protein KLH to serve as immunogen.

The inventor also provides a hybridoma cell strain secreting anti-VISTA protein molecules, wherein the cell strain is a mouse hybridoma cell strain 11A4, the cell strain is preserved in China general microbiological culture Collection center (CGMCC) at 9 and 17 days 2020, has the preservation number of CGMCC NO 20766 and is addressed to the institute of microbiology of China academy of sciences No.3 of North West Lu 1 of the rising area of Beijing.

The inventor also provides the application of the monoclonal antibody in the immune detection of VISTA protein.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor finally provides a VISTA protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 4; the amino acid sequence of the variable region of the monoclonal antibody light chain is the amino acid sequence shown in SEQ ID NO.5, and the anti-VISTA monoclonal antibody is the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, 269 th to 289 th amino acids at the tail end of the VISTA protein are selected as antigen peptides, the mouse is immunized by immunogen obtained after coupling KLH, and a mouse hybridoma cell strain 11A4 capable of efficiently secreting the anti-VISTA protein monoclonal antibody and the anti-VISTA protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing VISTA protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing immunohistochemical staining results for urothelial cancer; wherein the left part is VISTA secreted by 11A4, and the right part is commercially available VISTA.

FIG. 2 is a graph comparing immunohistochemical staining results for epithelioid angiosarcoma; wherein the left part is VISTA secreted by 11A4, and the right part is commercially available VISTA.

FIG. 3 is a graph comparing immunohistochemical staining results of placental trophoblasts; wherein the left part is VISTA secreted by 11A4, and the right part is commercially available VISTA.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

EXAMPLE 1 preparation of immunogen

First, immunogen preparation

Through sequence and secondary structure analysis according to the protein sequence with the accession number of Q9H7M9 in Uniprot, the molecular weight of VISTA protein with the total length of 311 amino acids is about 33 kDa. According to The parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of The predicted protein by an online server http:// www.cbs.dtu.dk/services/NetSurfP/and The results of analysis of its antigenicity index (Jameson BA, Wolf H. The antigenicity index: a novel homology for predicting antigenic definitions. composite biosci. 1988,4(1): 181-6), The amino acid sequence RQPSESGRHLLSEPSTPLSPC at position 269-.

Coupling and purification of polypeptide

The equine activated keyhole limpet hemocyanin kit from Thermo Scientific (cat # 77653) was selected and operated according to the protocol provided for the kit. For the polypeptide to be conjugated, the free thiol group in the polypeptide is first detected with Ellman's reagent (Thermo Scientific, cat # 22582): adding 100 μ L Ellman reagent stock solution into 96-well plate, adding 10 μ L polypeptide solution, measuring its ultraviolet absorption value with Nano Drop spectrophotometer at λ =412 nm, and performing the next step if OD value is greater than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide synthesis step for quality control. At the beginning of coupling, 200. mu.L of deionized water was added to each mcKLH package to prepare a 10mg/mL KLH solution, 2mg of hapten was dissolved in 500. mu.L of Imject EDC coupling buffer, 500. mu.L of polypeptide solution was added to 200. mu.L of carrier protein solution, 1mL of deionized water was added to one package of EDC (10 mg) and shaken slowly to dissolve completely, 50. mu.L of polypeptide solution was added to mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment to obtain VISTA-KLH.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptide of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 2 SPF-grade BALB/c female mice and 2 ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized and injected 6 spots per mouse at a dose of 60 μ g/mouse by abdominal subcutaneous injection. The booster was given every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30 μ g/mouse. Detecting the titer of multiple antibodies of the anti-immunogen in the serum of the mouse by indirect ELISA (wavelength of 450 nm) 7 days after the 3 rd boosting immunization, injecting the mouse with the highest titer into tail vein for impact immunization, and uniformly mixing the antigen with physiological saline at a dose of 50 mug/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0 (ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100 μ L of supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely changed and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). And taking 100 mul of supernatant after five days, carrying out third ELISA screening, and gradually transferring the positive clones to a 6-hole plate and a cell culture bottle for amplification culture and freezing storage.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

The coated goat anti-mouse IgG (Beijing Zhonghua Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l per well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 50 μ l per well containing 0.15% ABTS (Southern Biotech) and 0.03% H₂O₂The reaction was performed in the citric acid buffer (pH 4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is IgG_2aType murine monoclonal antibodies.

Second, determination of affinity constant

The VISTA polypeptide prepared in example 1 was coated at a concentration of 2 μ g/ml and 100 μ l/well overnight at 4 ℃ and washed 3 times with PBS-T. Adding 200 mul of blocking solution into each well, blocking for 2h at 37 ℃, and washing for 3 times by PBS-T. The monoclonal antibody purified in example 3 was purified from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 mul per well, incubation for 1h at 37 ℃ and PBS-T washing for 3 times. 100 μ l of each well containing 0.1% TMB (Sigma Co.) and 0.03% H₂O₂The citric acid-phosphoric acid buffer solution was developed for 10min, and 50. mu.l of 0.5M sulfuric acid solution was added to terminate the reaction. Using enzyme-linked immunosorbent assayThe absorbance at a wavelength of 450nm was measured. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 1.92 multiplied by 10 by using the following formula⁹。

Affinity constant ≈

Reaction specificity and application effect of monoclonal antibody

VISTA polypeptide prepared in example 1 was taken, and the recognition specificity of the monoclonal antibody of the present invention was examined by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 11A4 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequences of antibodies

Collecting fresh hybridoma cells, collecting supernatant, performing antigen binding property verification to confirm that the cell strain for cloning can indeed secrete required antibody, and centrifuging to collect 10⁶The hybridoma cells described above. Total RNA of hybridoma cells was extracted by Trizol method, and 9. mu.L of total RNA was added with 2.5. mu.L of oligo (dT) 12-18 primer (10 mM) and 5. mu.L of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA thus obtained was subjected to PCR amplification, and 25 p of each primer was added to a 50. mu.L reaction systemThe sequences of the primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the sequence of the murine monoclonal antibody primer in the book "recombinant antibody" (scientific Press, 2005) filed by Higgera.

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ L of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, the length of light chain (. kappa.light chain) was between 320-340bp and the length of heavy chain was between 340-370bp, and the region-specific product was recovered by gel cutting and cloned into T-vector or expression vector for sequencing.

Example 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody VISTA (11A 4) and the commercial antibody VISTA (D5L 5T) were tested simultaneously on a multiple tumor tissue chip of 45 tumor tissues and the results were compared.

The immunohistochemical results of VISTA were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-VISTA protein secreted by the 11A4 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the positive detection, the number of immunocyte staining cells and the staining intensity in 1 epithelioid angiosarcoma and 1 urothelial carcinoma are obviously higher than those of the commercial VISTA, which indicates that the sensitivity is higher and the false negative result is effectively avoided.

FIG. 1 is a graph showing a comparison of immunohistochemical staining results for urothelial cancer (the left is VISTA secreted from 11A4 cell line, and the right is VISTA commercially available).

FIG. 2 is a comparison of immunohistochemical staining results for epithelioid angiosarcoma (VISTA secreted from 11A4 cell line on the left, and VISTA on the right, which is commercially available).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (11A 4) and the commercial antibody (D5L 5T) were detected on the normal tissue chip synchronously, and the results of the negative and positive detection were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

FIG. 2 is a comparison of the results of immunohistochemical staining of placental trophoblasts (left is VISTA secreted by 11A4 cell line, right is commercially available VISTA).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims

1. The anti-VISTA protein monoclonal antibody is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, which specifically recognizes a VISTA protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG_2aSubtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20766.

6. A hybridoma cell strain secreting anti-VISTA protein molecules is a mouse hybridoma cell strain 11A4, is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation number of: CGMCC NO 20766.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a VISTA protein immunoassay reagent.

8. The use according to claim 7, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

9. A VISTA protein immunohistochemical detection reagent, characterized in that the immunohistochemical detection reagent contains the anti-VISTA protein monoclonal antibody of claim 1 as an active ingredient.