Monoclonal antibody against human MSH2 and preparation method and application thereof

Abstract

The application discloses a kind of anti-human MSH2 monoclonal antibody and its preparation method and application.The monoclonal antibody of anti-human MSH2 provided by the application includes the VHCDR1, VHCDR2 and VHCDR3 of amino acid sequence as shown in SEQ ID NO:1~3, and the VLCDR1, VLCDR2 and VLCDR3 of amino acid sequence as shown in SEQ ID NO:4~6.The monoclonal antibody of anti-human MSH2 of the application has good specificity and affinity, and can be applied to immunohistochemical detection.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody against human MSH2, its preparation method, and its application, belonging to the field of immunology technology. Background Technology

[0002] Microsatellite instability (MSI) refers to the shortening or lengthening of microsatellite fragments caused by the increase or loss of repetitive sequences in the genome due to gene replication errors. MSH2 is involved in the initial recognition of nucleotides during mismatch repair after replication. Therefore, the loss of MSH2 function leads to the accumulation of replication errors, which may in turn lead to the occurrence of multiple mutations required for multi-stage carcinogenesis.

[0003] MSH2 has been reported to be expressed in the nuclei of cells in various tissues, including the thyroid gland, heart, smooth muscle, and germinal centers of lymphoid follicles. Mutations in the MSH2 gene are associated with microsatellite instability and some cancers; they contribute to the development of sporadic colorectal cancer, and MSH2 mutations cause 50% of hereditary nonpolyposis colorectal cancer (HNPCC). Therefore, the development of MSH2 monoclonal antibodies is of great significance for the pathological diagnosis and drug development of this type of tumor.

[0004] In the development of MSH2 monoclonal antibodies for immunohistochemical (IHC) detection, according to the evaluation results of NordiQC (currently the highest quality control committee in the pathology industry worldwide), regarding MSH2 monoclonal antibodies, including imported products with clone numbers 25D12, BSR77, FE11, G219-1129, GB12, and RED2, FE11 is the most widely used and its efficacy is slightly better than other clone numbers, but its evaluation score is still not high. Furthermore, imported products are expensive and have long lead times, which is detrimental to the routine pathological diagnosis work in this field in China. Therefore, developing a clinically applicable anti-MSH2 monoclonal antibody is of great significance in order to obtain an anti-MSH2 monoclonal antibody with efficacy comparable to or superior to imported reagents, while simultaneously reducing the burden on patients and lowering treatment costs. Summary of the Invention

[0005] The first objective of this invention is to provide a monoclonal antibody against human MSH2 that is stable and has good specificity and affinity.

[0006] A second objective of this invention is to provide the application of the above-mentioned monoclonal antibody in the preparation of MSH2 in vitro detection reagents or kits.

[0007] A third objective of this invention is to provide an immunohistochemical detection reagent or kit containing the above-mentioned monoclonal antibody.

[0008] A fourth objective of this invention is to provide an application of the above-described monoclonal antibody in immunohistochemistry.

[0009] The fifth objective of this invention is to provide a method for preparing the above-mentioned monoclonal antibody.

[0010] A sixth object of the present invention is to provide a nucleic acid molecule comprising the gene encoding the monoclonal antibody described above.

[0011] A seventh objective of the present invention is to provide an expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the above-mentioned nucleic acid molecules.

[0012] The eighth object of the present invention is to provide the use of the above-mentioned nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria in the preparation of anti-MSH2 monoclonal antibodies.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A monoclonal antibody against human MSH2, comprising VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0015] The monoclonal antibody against human MSH2 provided by this invention was prepared using the N-terminal 2-515 amino acid region of the P43246 protein sequence published by Uniprot as the antigen. The monoclonal antibody against human MSH2 of this invention is stable and exhibits good specificity and sensitivity.

[0016] Preferably, the heavy chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO: 7, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.

[0017] Application of anti-human MSH2 monoclonal antibodies in the preparation of in vitro detection reagents or kits for MSH2.

[0018] This monoclonal antibody can be used to prepare in vitro detection reagents or kits for MSH2, which can locate MSH2 protein in tissues and cells, and also detect the expression of MSH2 in tissues and cells.

[0019] An immunohistochemical detection reagent or kit containing a monoclonal antibody against human MSH2.

[0020] This immunohistochemical reagent or kit can be used for in vitro immunoassay of MSH2 expression in tissue cells.

[0021] Application of anti-human MSH monoclonal antibody in immunohistochemistry.

[0022] The monoclonal antibody against human MSH2 of the present invention has better specificity and sensitivity than imported antibodies and can be widely used in immunohistochemical detection.

[0023] A method for preparing a monoclonal antibody against human MSH2 includes the following steps:

[0024] (1) Rabbits were immunized with MSH2 immunogen, rabbit peripheral blood was collected, rabbit peripheral blood mononuclear cells were collected, and antigen-specific B lymphocytes were isolated.

[0025] (2) For the antigen-specific B lymphocytes obtained in step (1), positive cell pores are selected through culture and identification, and then antibody heavy chain and light chain genes are obtained. The target genes of antibody heavy chain and light chain are amplified, and rabbit monoclonal antibodies are obtained through recombination and expression.

[0026] In the method for preparing the monoclonal antibody against human MSH2 of the present invention, the antibody nucleotide sequence is known, the antibody gene can be preserved for a long time, the prepared antibody is stable, and has good specificity and affinity.

[0027] A nucleic acid molecule containing a gene sequence encoding a monoclonal antibody against human MSH2.

[0028] This nucleic acid molecule can be obtained through genetic engineering recombination technology or chemical synthesis methods. Those skilled in the art will readily understand that the variant sequences of the heavy chain variable region nucleotide sequence and / or light chain variable region nucleotide sequence obtained by mutation of the above-mentioned nucleic acid molecule provided in this invention through the addition, deletion, substitution, modification, etc., will still retain the ability to specifically bind to the MSH2 protein when the encoded amino acid sequences form single-chain antibodies, chimeric monoclonal antibodies, modified monoclonal antibodies, or other forms of monoclonal antibodies or antibody fragments.

[0029] Preferably, the nucleotide sequence of the heavy chain variable region gene of the anti-human MSH2 monoclonal antibody is shown in SEQ ID NO: 9; and the nucleotide sequence of the light chain variable region gene of the anti-human MSH2 monoclonal antibody is shown in SEQ ID NO: 10.

[0030] An expression cassette, recombinant vector, recombinant cell, and recombinant bacteria comprising the aforementioned nucleic acid molecules.

[0031] Preferably, the recombinant expression vector is selected from prokaryotic or eukaryotic expression vectors; more specifically, the recombinant expression vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0032] Preferably, the expression system is a bacterial, yeast, filamentous fungus, mammalian cell, insect cell, plant cell, or cell-free expression system.

[0033] The application of the above-mentioned nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, and recombinant bacteria in the preparation of monoclonal antibodies against human MSH2.

[0034] This invention provides nucleic acid molecules, expression cassettes, expression vectors, recombinant cells, and recombinant bacteria containing the variable region amino acid sequences of the heavy and light chains of a monoclonal antibody against human MSH2. Based on this, the monoclonal antibody against human MSH2 of this invention can be obtained using conventional genetic engineering methods. Attached Figure Description

[0035] Figure 1 The immunohistochemical results (200×) of the 2G5 antibody in colorectal cancer tissue in Example 4 of this invention;

[0036] Figure 2 The immunohistochemical results (200×) of FE11 antibody in colorectal cancer tissue in Example 4 of this invention;

[0037] Figure 3 The immunohistochemical results (200×) of the 2G5 antibody in gastric cancer tissue in Example 4 of this invention;

[0038] Figure 4 The immunohistochemical results (200×) of FE11 antibody in gastric cancer tissue in Example 4 of this invention. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0040] Example 1: Monoclonal Antibody Against Human MSH2

[0041] The monoclonal antibody against human MSH2 in this embodiment has the amino acid sequence of its heavy chain variable region as shown in SEQ ID NO: 7, and the amino acid sequence of its light chain variable region as shown in SEQ ID NO: 8. The heavy chain variable region contains VHCDR1, VHCDR2, and VHCDR3 as shown in SEQ ID NO: 1–3, and the light chain variable region contains VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO: 4–6.

[0042] Example 2: Preparation method of anti-human MSH2 monoclonal antibody

[0043] The method for preparing anti-human MSH2 monoclonal antibody in this embodiment involves immunizing rabbits with the MSH2 immunogen, collecting rabbit peripheral blood, collecting rabbit peripheral blood mononuclear cells, and isolating antigen-specific B lymphocytes. The obtained antigen-specific B lymphocytes are cultured, identified, and screened to select positive cell wells, from which the antibody heavy chain and light chain genes are obtained. The target genes for the antibody heavy chain and light chain are amplified, and the rabbit-derived monoclonal antibody is obtained through recombination and expression. The specific implementation steps are as follows:

[0044] 2.1 Acquisition of MSH2 antigen

[0045] Based on the P43246 protein sequence published by Uniprot, the N-terminal 2-515 amino acid region was selected as the antigen sequence, whose amino acid sequence is shown in SEQ ID NO: 11. After codon optimization of its nucleotide sequence, the optimized nucleic acid sequence was artificially synthesized and constructed into the pET28a vector, which was then transformed into E. coli for protein expression. The target protein with a purity of up to 90% was obtained after purification by nickel column affinity chromatography.

[0046] 2.2 Immunized Animals

[0047] To further improve the animal's immune response, CSF2 cytokine was added as an immune chaperone. CSF2 was a self-constructed and expressed protein, obtained through the following methods: The natural CSF2 protein gene containing a promoter and terminator was obtained from a database; the CSF2 gene was inserted into mammalian expression vectors such as pcDNA3.1 using traditional vector construction methods; the successfully constructed vector was transfected into a rabbit-derived mammalian expression system without relevant expression, and the expression level of CSF2 protein was detected using routine protein assays; the CSF2 protein was purified using immunoaffinity chromatography.

[0048] Immunogen and immune chaperone (CSF2 protein) were added to a water-soluble adjuvant to obtain an immunoassay reagent. The concentration of the immune chaperone in the immunoassay reagent was controlled at 50 μg / mL, and the immunogen dose was 100 μg / rabbit. New Zealand white rabbits were then immunized via intramuscular injection in the leg, for a total of three immunizations, each 21 days apart, with the same dose for each immunization. On day 14 after the third immunization, blood was collected from the marginal ear vein to assess the immunization effect. Once the expected efficacy was achieved, the rabbits underwent a pulse immunization with MSH2 immunogen, and 10 mL of blood was collected from the marginal ear vein on day four.

[0049] 2.3 Rabbit peripheral blood collection and PBMC isolation, B lymphocyte sorting and culture

[0050] PBMCs (peripheral blood mononuclear cells, which can be obtained using a commercially available rabbit peripheral blood lymphocyte isolation kit) were isolated from the peripheral blood of rabbits after shock immunization. Subsequently, the cells were screened again using one or more antibodies against T lymphocyte surface markers such as CD4, CD8, CD14, CD28, and CD80 to remove T cells, monocytes, etc.

[0051] Activated B lymphocytes were then selected using FITC-labeled goat anti-rabbit IgG.

[0052] Then, using a commercially available biotin labeling kit, the immunogen MSH2 was labeled according to the instructions. This was then combined with a commercially available avidin-conjugated fluorescein (PE, APC, PerCP, PE-Cy7, APC-Cy7, etc.) reagent and incubated at 37°C for 1 hour (with agitation several times during incubation) to obtain the antigen-biotin-avidin screening reagent. When using, adjust the dosage according to the cell number (1×10⁻⁶). 6 Add 5 μL of cells to the B lymphocytes and perform positive screening again to obtain antigen-specific B lymphocytes.

[0053] The sorted B lymphocytes were seeded into 96-well plates with 1-2 cells per well, which were pre-filled with feeder cells. One or more cytokines from IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-β, and SAC were added for co-culture.

[0054] 2.4 ELISA detection of lymphocyte culture supernatant

[0055] After culturing for 7-12 days, the cell supernatant was collected for ELISA detection. The cells were coated with the immunogen MSH2, and serum collected before rabbit immunization was used as a negative control well. A positive result was defined as an OD450 reading 2.1 times greater than that of the negative control well. The selected positive wells were then selected.

[0056] 2.5. cDNA acquisition, antibody heavy and light chain gene amplification and sequencing

[0057] Cells from the selected positive cell wells were lysed, and total RNA was extracted to obtain cDNA via RT-PCR. Antibody heavy and light chain gene bands from the cells in the wells were amplified using conventional PCR with one primer pair for each of the heavy and light chains. The primer sequences are shown in SEQ ID NO: 12–15. In the primer sequences, R and S are degenerate codons. Because the antibody sequence is a variable region, degenerate primers were used to retrieve the antibody gene. R represents A / G, and S represents G / C. The obtained bands were recovered from the gel to obtain the antibody heavy and light chain gene products. A portion of the obtained products was sequenced. The primer sequences used for sequencing are shown in SEQ ID NO: 16–17.

[0058] 2.6 Constructing recombinant plasmids for heavy chain antibodies and light chain antibodies

[0059] After analyzing the correctly sequenced antibody heavy and light chain gene sequences, the variable region sequence of the heavy and light chains was extracted and recombined with the self-modified vectors pRCH and pRCK containing the constant region sequence of the heavy and light chain antibody gene.

[0060] DH5α strain containing pRCH and pRCK plasmids were cultured on a large scale, and plasmids were extracted and double-digested (EcoRI and HindIII). Plasmid extraction was performed according to the Axygen plasmid extraction kit instructions. The double-digestion reaction system was: 2 μg pRCH or pRCK plasmid, 2 μL EcoRI, 2 μL HindIII, 5 μL buffer, and 40 μL ddH2O; the digestion conditions were 37°C water bath for 2 h. The digested system was subjected to agarose gel electrophoresis, and the double-digested plasmid fragments were recovered from the gel, following the Axygen gel recovery kit instructions.

[0061] The antibody heavy and light chain variable region gene fragment was obtained by PCR. The upstream and downstream primer sequences are shown in SEQ ID NO: 18-21. The PCR amplification cycle settings were: I: 98℃ 3 min; II: 98℃ 10 s; III: 56℃ 10 s; IV: 72℃ 10 s; II-IV: 30 cycles; V: 72℃ 5 min. The target band was obtained by 1.5% agarose gel electrophoresis, and the target fragment was excised and recovered. This procedure was performed according to the Axygen gel recovery kit instructions.

[0062] Homologous recombination of the target gene fragment and the double-digested plasmid fragment was performed using a seamless cloning kit. This step was performed in accordance with the instructions of the nearshore seamless cloning kit (catalog number: NR005-01A).

[0063] After seamless recombination, the cells were transformed into DH5α competent cells and screened using ampicillin-resistant LB plates. The cells were cultured at 37°C for 16-18 hours, and five single clones from each plate were selected for gene sequencing. Colonies with correct sequencing were expanded and plasmids were extracted.

[0064] 2.7 Plasmid transfection

[0065] High-concentration, high-purity antibody heavy and light chain recombinant plasmids obtained after expanded culture were transfected into CHO / HEK293 cells according to the PEI transfection reagent instructions. The antibody heavy and light chains were pre-mixed at a molar ratio of 1:2, and the plasmid was diluted with CHO / HEK293 cell basal medium. Simultaneously, an equal volume of the medium was used to dilute PEI (plasmid:PEI = 1:3, w / w). The mixture was then transfected into CHO / HEK293 cells cultured in the logarithmic growth phase. Forty-eight hours after transfection, the antibody titer secreted in the supernatant was detected using an indirect ELISA method to identify cell lines with relatively high expression levels.

[0066] 2.8. Antibody engineered expression and purification

[0067] The selected cell line was expanded to a volume of 100 mL, and the cell growth state was adjusted to the logarithmic growth phase. Plasmids were transfected, and feed was added every other day. The cell supernatant was harvested on day 6 post-transfection. The supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a Protein G column. The antibodies obtained from the binding adsorption were eluted with citrate buffer at pH 6.0, and the eluent was collected. The pH was rapidly neutralized to between 7.2 and 7.4 using Tris-HCl solution at pH 8.8. The purified antibody was then concentrated by ultrafiltration and centrifugation to a concentration above 1 mg / mL.

[0068] Example 3 Immunohistochemical detection reagents or kits

[0069] The immunohistochemical detection reagent used in this embodiment is the MSH2 immunohistochemical detection reagent, which is the working solution of the MSH2 monoclonal antibody in Example 1. The working concentration of the antibody is 0.5 ug / mL, the solvent is TBS buffer, and protective protein BSA (bovine serum albumin, content is 10 mg / mL) is added. Preservatives Proclin-300 and Proclin-950 are added (both added at a ratio of 1:1000 (V / V)).

[0070] Based on the monoclonal antibody working solution in the examples, an immunohistochemical detection kit can be constructed by adding supporting reagents such as antigen EDTA retrieval solution, hydrogen peroxide blocking solution, horseradish peroxidase-labeled goat anti-mouse / rabbit secondary antibody, DAB and its buffer, and hematoxylin.

[0071] Example 4: Application of anti-human MSH2 monoclonal antibodies in immunohistochemistry

[0072] This embodiment demonstrates the application of the anti-human MSH2 monoclonal antibody in immunohistochemistry. Using the immunohistochemical detection kit from Example 3, MSH2 protein was detected in colorectal cancer and gastric cancer tissues, respectively. The specific operating steps are as follows:

[0073] 4.1 Application in colorectal cancer tissue

[0074] The immunohistochemical detection kit from Example 3 was used to stain colorectal cancer tissue. The staining process is as follows:

[0075] 1. Paraffin-embedded tissue sections of colorectal cancer, 3µm / section, baked at 65℃ for 2 hours;

[0076] 2. Dewaxing and hydration: Paraffin sections are treated as follows: xylene 15min - xylene 15min - anhydrous ethanol 5min - anhydrous ethanol 5min - 90% ethanol 5min - 80% ethanol 5min - 70% ethanol 5min, and then soaked in purified water for 5min.

[0077] 3. Antigen retrieval: After heating and boiling the EDTA antigen retrieval solution at pH 9.0, place the tissue slide in the solution, adjust to a gentle boiling mode, and time for 20 minutes. After cooling naturally for 5 minutes, rinse with running water to cool to room temperature, remove the slide, soak in purified water for 5 minutes, and then rinse and soak in TBST for 5 minutes.

[0078] 4. Add peroxidase blocking agent: 100uL / sheet, incubate at room temperature for 5min, rinse and soak with TBST for 5min / time, for a total of 2 times.

[0079] 5. Primary antibody incubation: Add the above antibody working solution, 100uL / sheet, incubate at 37℃ for 30min, rinse and soak with TBST for 5min / time, for a total of 2 times.

[0080] 6. Secondary antibody incubation: Add horseradish peroxidase-labeled goat anti-mouse / rabbit secondary antibody, 100uL / sheet, incubate at room temperature for 30min, rinse and soak with TBST for 5min / time, for a total of 2 times.

[0081] 7. Color development: Add 100uL / sheet of DAB color development solution, incubate at room temperature for 4min, and soak twice in purified water for 5min each time.

[0082] 8. Counterstaining: Add 100 μL / sheet of hematoxylin staining solution, incubate at room temperature for 4 min, and rinse thoroughly with purified water.

[0083] 9. Dehydrated and transparent: Dehydrated with conventional gradient ethanol, and transparent with xylene.

[0084] 10. Mount the slide with neutral resin and observe under a microscope.

[0085] The staining results of antibody 2G5 prepared by the above examples and imported antibody FE11 are as follows: Figure 1 and Figure 2 As shown. By Figure 1 and Figure 2 The comparison results show that, compared with the control antibody FE11, 2G5 has better specificity and affinity, and the antibody concentration of 0.5ug / mL (the antibody concentration of FE11 is about 1ug / mL) can achieve a strong staining effect and a better background.

[0086] 4.2 Application in gastric cancer tissue

[0087] Gastric cancer tissue was stained using the immunohistochemical detection kit described in Example 3. The staining process was as described in section 4.1 of Example 4. The staining results of 2G5 and the imported antibody FE11 are as follows: Figure 3 and Figure 4 As shown.

[0088] Depend on Figure 3 and Figure 4 The comparison results show that, compared with the control antibody FE11, 2G5 has better specificity and affinity, and the antibody concentration of 0.5ug / mL (the antibody concentration of FE11 is about 1ug / mL) can achieve a strong staining effect and a better background.

[0089] 4.3 Consistency Evaluation

[0090] Following the staining process described in the above embodiments, immunohistochemical detection and evaluation were performed on multiple tissues using control antibody FE11 and antibody 2G5. The results are shown in Table 1 below. The evaluated tissues included colorectal cancer tissue, gastric cancer tissue, lung cancer tissue, tonsils, appendix, placenta, moderately differentiated hepatocellular carcinoma of the liver, serous carcinoma of the ovary, cervical cancer, pancreas, thyroid follicles, pheochromocytoma, malignant melanoma, urothelial carcinoma, glioblastoma, thymoma, normal kidney, and normal colon, totaling 134 tissues.

[0091] Table 1. Statistical analysis of tissue evaluation of control antibody FE11 and antibody 2G5

[0092]

[0093]

[0094] As shown in Table 1, there was no difference between antibody 2G5 in the examples and control antibody FE11 in terms of the evaluation of immunohistochemical detection results.

[0095] Example 5: Nucleic acid molecule of anti-human MSH2 monoclonal antibody gene sequence

[0096] The nucleic acid molecule of the monoclonal antibody gene sequence against human MSH2 in this embodiment has the nucleotide sequence of the heavy chain variable region as shown in SEQ ID NO: 9 and the nucleotide sequence of the light chain variable region as shown in SEQ ID NO: 10.

[0097] In summary, the anti-human MSH2 monoclonal antibody obtained by this invention can specifically bind to MSH2 in tissue cells and is correctly localized in tissues, making it suitable for pathological diagnosis and scientific research related to MSH2. Compared with traditional monoclonal antibody preparation methods, the method for preparing the anti-human MSH2 monoclonal antibody of this invention has the advantages of known antibody sequences, long-term preservation of antibody genes, and stable antibody properties.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against human MSH2, characterized in that: It contains the amino acid sequences VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO: 1~3, and the amino acid sequences VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO: 4~6.

2. The monoclonal antibody against human MSH2 according to claim 1, characterized in that: The monoclonal antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

8.

3. The use of a monoclonal antibody as described in claim 1 or 2 in the preparation of an in vitro detection reagent or kit for MSH2.

4. An immunohistochemical detection reagent or kit comprising the monoclonal antibody as described in claim 1 or 2.

5. A nucleic acid molecule comprising a gene sequence encoding a monoclonal antibody against human MSH2 as described in claim 1 or 2.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence of the heavy chain variable region gene of the anti-human MSH2 monoclonal antibody is shown in SEQ ID NO: 9; the nucleotide sequence of the light chain variable region gene of the anti-human MSH2 monoclonal antibody is shown in SEQ ID NO:

10.

7. An expression cassette, recombinant vector, recombinant cell, or recombinant bacterium comprising the nucleic acid molecule of claim 5.

8. The use of a nucleic acid molecule as described in claim 5 or 6, or an expression cassette, recombinant vector, recombinant cell, or recombinant bacterium as described in claim 7, in the preparation of an anti-human MSH2 monoclonal antibody.

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