CN113234158B

CN113234158B - anti-TIM 3 protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN113234158B
Application number: CN202110517834.XA
Authority: CN
Inventors: 杨清海; 陈惠玲; 王小亚; 周洪辉
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-05-13
Anticipated expiration: 2041-05-12
Also published as: CN113234158A

Abstract

The invention relates to a monoclonal antibody capable of recognizing human TIM3 antigen, a secretory cell strain, a preparation method and application thereof in immunoassay. The technical scheme chemically synthesizes amino acid at the C-terminal position 178-201 of the TIM3 protein as antigen peptide, and obtains immunogen after coupling KLH. The TIM3-KLH protein is used for immunizing a mouse, and a mouse hybridoma cell strain 7H2 capable of efficiently secreting the anti-TIM 3 protein monoclonal antibody and the anti-TIM 3 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 TIM3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-TIM 3 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-TIM 3 protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

Malignant tumors seriously affect human health, and the current tumor treatment means include radiotherapy, chemotherapy and molecular targeting treatment. The killing process of the cell immune system to the tumor cells comprises the recognition of the tumor cells, the presentation of tumor antigens to T cells, the activation of the T cells and the specific killing of the tumor cells. In this process, the activation of T cells requires the coordinated stimulation signals, and these signals require the regulation of "immune checkpoint", which is widely studied in T cell immunoglobulin mucin 3(TIM3), cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death factor-1 (PD-1) and its ligand (PD-L1), etc., and they play an important role in the immune regulation of tumor microenvironment.

TIM3(T cell immunoglobulin mucin 3, T cell immunoglobulin and mucin-3) was first discovered in 2002, TIM3 is a member of the TIM family, a type I membrane protein consisting of 301 amino acids. The common structure of TIM molecules includes the immunoglobulin N-terminal variable region, mucin domain, transmembrane region and intracellular region. The extracellular portion of which comprises a cysteine-rich immunoglobulin-like domain and a mucin domain (the mucin domain is rich in threonine, serine and proline), a highly conserved unique cleft consisting of 4 cysteines in the immunoglobulin variable domain, distinct from other immunoglobulin superfamily members, which can bind to Phosphatidylserine (PS). TIM3 is selectively expressed on IFN- γ secreting T helper cells (Th1 and Th17), T regulatory cells (tregs), Dendritic Cells (DCs), monocytes, mast cells, NK cells, Tumor Infiltrating Lymphocytes (TILs), and also on tumor cells, such as melanoma, gastric cancer, B cell lymphoma.

Disclosure of Invention

The inventor provides an anti-TIM 3 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as 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 TIM3 protein.

Further, the monoclonal antibody is mouse IgG₁Subtype monoclonal antibodies.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20763.

The inventor also provides a preparation method of the monoclonal antibody against the TIM3 protein, which selects an amino acid sequence (178 th to 201 th) shown as SEQ ID No.1 in the TIM3 protein, adds a cysteine at the carboxyl terminal thereof, and then couples the amino acid sequence with a carrier protein KLH to be used as immunogen.

The inventor also provides a hybridoma cell strain secreting anti-TIM 3 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 7H2, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 20763) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20763, and the cell strain is the institute of microbiology of China academy of sciences No.3 of the North West Lu No.1 Hopkins of the Korean-Yang district in Beijing.

The inventor also provides the application of the monoclonal antibody in the immunoassay of the TIM3 protein.

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

The inventor finally provides a TIM3 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 light chain variable region is the amino acid sequence shown in SEQ ID NO.5, and the anti-TIM 3 protein monoclonal antibody is used as the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, amino acids 178-201 of the C-terminal of the TIM3 protein are selected as antigen peptides, an immunogen obtained after coupling KLH is used for immunizing a mouse, and a mouse hybridoma cell strain 7H2 capable of efficiently secreting an anti-TIM 3 protein monoclonal antibody and an anti-TIM 3 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 TIM3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a comparison of immunohistochemical staining of tonsils (left 7H2 secreted TIM3, right commercial TIM 3).

FIG. 2 is a comparison of the results of immunohistochemical staining of the appendix (left for TIM3 secreted by 7H2 and right for commercially available TIM 3).

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

Sequence and secondary structure analysis is carried out according to the protein sequence with the accession number of Q8TDQ0 in Uniprot, and the molecular weight of TIM3 protein with the total length of 301 amino acids is about 33 kDa. According to the parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of the protein predicted by the online server http:// www.cbs.dtu.dk/services/NetSurfP/and by the results of analysis of its antigenicity index (Jameson BA, Wolf H. the antigenicity index: a novel homology for predicting antigenic definitions. composite applied biosci.1988,4(1):181-6.), the amino acid sequence SEQ ID NO.1 at position 178 and 201 is selected as the antigenic peptide for chemical coupling, and a cysteine is added at the carboxyl end of the polypeptide to provide thiol coupling.

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 mu L Ellman reagent stock solution into a 96-well plate, adding 10 mu L polypeptide solution, measuring the ultraviolet absorption value of the solution by a Nano Drop spectrophotometer at the lambda of 412nm, and performing the next step if the OD value is more 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. To begin conjugation, 200. mu.L of deionized water was added to each mcKLH package to make up a 10mg/mL solution of KLH, 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 (10mg) 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 give TIM 3-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 ICR mice (purchased from Beijing Wintotonghua laboratory animal technology Co., Ltd.) were immunized and injected ventrally at 6-point per mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/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 1500rpm 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 1000rpm 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 10 XHAT (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.

Thirdly, cloning and ELISA screening positive hybridoma cell

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. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced 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). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen. 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

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/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 2000 by 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. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), 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 IgG₁Type murine monoclonal antibodies.

Second, determination of affinity constant

The TIM3 polypeptide prepared in example 1 was taken at a coating concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with 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 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. 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 3.84 × 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The TIM3 polypeptide prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and subjected to 12% polyacrylamide gel electrophoresis. 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 7H2 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

The cultured fresh hybridoma cells were taken and the supernatant was subjected to antigen binding property verification to confirm that the cell line used for cloning was indeed secreting the desired oneAntibody, after confirmation of results, centrifugation 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 (10mM) 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 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized based on the sequence of the murine monoclonal antibody primer as described in "recombinant antibody" by Hippo Seiyaku (science publishers, 2005 publications).

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. Tissue chip detection results:

the antibody TIM3(7H2) and the commercial antibody TIM3(D5D5R) were tested simultaneously on tonsil and appendix tissue chips and the results were compared.

Immunohistochemical results for TIM3 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the results show that the monoclonal antibody of the anti-TIM 3 protein secreted by the 7H2 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In a positive test, the number of stained cells and staining intensity of TIM3 antibody secreted by 7H2 were significantly higher than those of commercially available TIM 3; the TIM3 antibody secreted by 7H2 is more sensitive, and false negative results are effectively avoided.

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 (7H2) and the commercial antibody (E3) were detected on the normal tissue chip synchronously, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

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. An anti-TIM 3 protein monoclonal antibody, which 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, wherein the monoclonal antibody specifically recognizes TIM3 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG₁Subtype monoclonal antibody。

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20763.

6. A hybridoma cell strain secreting anti-TIM 3 protein molecules is a mouse hybridoma cell strain 7H2, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20763.

7. Use of a monoclonal antibody according to any one of claims 1-5 in the preparation of a TIM3 protein immunoassay reagent.

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

9. An immunohistochemical detection reagent for TIM3 protein, comprising the anti-TIM 3 monoclonal antibody according to claim 1 as an active ingredient.