Anti-tumor antigen BAP31 monoclonal antibody, Cy5-BAP31 directly labeled monoclonal antibody and its application

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses an anti-tumor antigen BAP31 monoclonal antibody, a Cy5-BAP31 directly labeled monoclonal antibody and its application. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.3. The nucleotide sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.4. The anti-BAP31 monoclonal antibody provided by the present invention can specifically recognize the BAP31 molecule, inhibit the subcutaneous tumor formation ability and in vivo metastasis ability of liver cancer cells, and significantly prolong the survival of tumor-bearing nude mice.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-tumor antigen BAP31 monoclonal antibody, a Cy5-BAP31 directly labeled monoclonal antibody and applications thereof. Background Art

[0002] Cancer is a major public health and economic issue in the 21st century. Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, typically develops from chronic liver disease. Its causative factors include hepatitis B / C virus infection, non-alcoholic fatty liver disease, alcoholic hepatitis, smoking, alcoholism, obesity, diabetes, iron overload, and various dietary exposures. Because early symptoms of HCC are not obvious, over 80% of HCC patients are beyond effective treatment at the time of diagnosis. Therefore, the early diagnosis and treatment of HCC remain a challenge for clinicians and researchers, and new diagnostic markers and effective treatments require continuous exploration and trial.

[0003] BAP31 is located in the endoplasmic reticulum and consists of an N-terminal membrane-binding domain composed of three transmembrane helices and a C-terminal cytoplasmic region. The cytoplasmic region, which comprises approximately 50% of the entire protein and forms a multi-coiled-coil structure, also contains cleavage sites for caspase-1 and -8. BAP31 is a molecular chaperone protein that plays an important role in the synthesis, error correction, and transport of many membrane proteins. Furthermore, BAP31 is involved in regulating Bcl-2 / Bcl-XL-mediated apoptosis. Our research group, using a unique method for screening tumor / testis antigens using spermatogenic cell-specific monoclonal antibodies, identified BAP31 as a novel tumor / testis antigen. Further analysis of its expression distribution revealed for the first time that BAP31 is absent or only weakly expressed in normal human tissues but highly expressed in various tumor tissues, including liver cancer, cervical cancer, and lung cancer. The expression rate in liver cancer tissue was particularly high, at 72.6%. Further research revealed that BAP31 is highly expressed in liver cancer and positively correlated with tumor size, lymph node metastasis, and clinical stage. Cell and animal experiments also confirmed that BAP31 promotes liver cancer cell proliferation and monoclonal formation. These findings suggest that BAP31 may be a novel liver cancer-associated antigen that plays an important role in liver cancer progression.

[0004] Therefore, whether or not a monoclonal antibody that specifically binds to BAP31 can be developed is of great significance for the preparation of liver cancer diagnostic products and the treatment of liver cancer. Summary of the Invention

[0005] To obtain a new product for diagnosing and / or treating liver cancer, the present invention provides an anti-tumor antigen BAP31 monoclonal antibody, a Cy5-BAP31 directly-labeled monoclonal antibody, and their uses. The anti-BAP31 monoclonal antibody FMU-BAP31-No.1 provided by the present invention can specifically recognize the BAP31 molecule, inhibit the subcutaneous tumor formation and in vivo metastasis ability of liver cancer cells, and significantly prolong the survival of tumor-bearing nude mice. At therapeutic doses, the BAP31 monoclonal antibody has no effect on the heart, liver, lung, and kidney tissues of nude mice.

[0006] The present invention provides an anti-tumor antigen BAP31 monoclonal antibody, the monoclonal antibody being FMU-BAP31-No.1, comprising a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of the monoclonal antibody FMU-BAP31-No.1 is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.3. The nucleotide sequence of the light chain variable region of the monoclonal antibody FMU-BAP31-No.1 is shown in SEQ ID NO.2, and the amino acid sequence thereof is shown in SEQ ID NO.4.

[0007] Furthermore, the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown as SEQ ID NO.5; the amino acid sequence of CDR2 is shown as SEQ ID NO.6; and the amino acid sequence of CDR3 is shown as SEQ ID NO.7.

[0008] The present invention also provides a Cy5-BAP31 directly labeled monoclonal antibody, which is obtained by labeling the monoclonal antibody FMU-BAP31-No.1 with Cy5.

[0009] The present invention also provides a use of the Cy5-BAP31 directly labeled monoclonal antibody in preparing a BAP31 detection reagent.

[0010] The present invention also provides a use of the anti-tumor antigen BAP31 monoclonal antibody in the preparation of an immunoassay tool.

[0011] Furthermore, the immunoassay tool is a kit, a chip or a test paper.

[0012] Furthermore, the kit is an immunohistochemistry detection kit.

[0013] The present invention also provides a use of the anti-tumor antigen BAP31 monoclonal antibody in preparing a preparation for diagnosing and / or treating liver cancer.

[0014] Furthermore, the preparation is a medicine; the medicine has the following effects:

[0015] Inhibit the proliferation of liver cancer cells;

[0016] Inhibits the migration ability of liver cancer cell lines;

[0017] Inhibits subcutaneous tumor formation of liver cancer cell lines in nude mice;

[0018] Inhibit the metastatic ability of liver cancer cells in vivo;

[0019] Improve the survival of nude mice.

[0020] Furthermore, the liver cancer cells are Hep3b or MHCC97h.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The anti-BAP31 monoclonal antibody FMU-BAP31-No.1 provided by the present invention can specifically recognize the BAP31 molecule. In vitro and in vivo experiments have confirmed that the anti-BAP31 monoclonal antibody FMU-BAP31-No.1 can inhibit the subcutaneous tumor formation and in vivo metastasis ability of liver cancer cells, significantly prolong the survival of tumor-bearing nude mice, and at therapeutic doses, the BAP31 monoclonal antibody has no effect on the heart, liver, lung, and kidney tissues of nude mice.

[0023] 2. The present invention cloned the heavy chain variable region gene and amino acid sequence of the anti-BAP31 monoclonal antibody FMU-BAP31-No.1, and sequence analysis confirmed the uniqueness of the antibody sequence.

[0024] 3. Analyze and obtain the CDR region of the heavy chain variable region, and on this basis provide support for the construction of candidate targeted drugs for liver cancer and different forms of genetically engineered antibodies against BAP31. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The monoclonal antibody FMU-BAP31-No.1 prepared by the present invention and the commercial BAP31 rabbit polyclonal antibody were used for Western blot detection of BAP31 molecules. In the figure, the homemade BAP31 mouse monoclonal antibody is the monoclonal antibody FMU-BAP31-No.1.

[0027] Figure 2The flow cytometry results of the Cy5-BAP31 directly labeled monoclonal antibody prepared by the present invention binding to the natural BAP31 molecules on the surface of Hep3b and MHCC97h liver cancer cell lines;

[0028] In the figure, A is the flow cytometry result of Hep3b liver cancer cell line;

[0029] B is the flow cytometry result of the binding of Cy5-BAP31 directly labeled monoclonal antibody to the natural BAP31 molecule on the surface of Hep3b liver cancer cell line;

[0030] C is the flow cytometry result of MHCC97h liver cancer cell line;

[0031] D is the flow cytometry detection result of the Cy5-BAP31 directly labeled monoclonal antibody binding to the natural BAP31 molecules on the surface of the MHCC97h liver cancer cell line.

[0032] Figure 3 Immunohistochemical staining of samples from patients with liver cancer at different clinical stages using the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention;

[0033] In the figure, a is the immunohistochemical staining of the monoclonal antibody FMU-BAP31-No.1 in the adjacent tissues of the cancer, and a1 is a local magnified view of a;

[0034] b is the immunohistochemical staining of the monoclonal antibody FMU-BAP31-No.1 in liver cancer tissues of patients with stage I liver cancer; b1 is a partial magnified view of b;

[0035] c is the immunohistochemical staining of the monoclonal antibody FMU-BAP31-No.1 in liver cancer tissues of patients with stage II liver cancer; c1 is a partial magnified view of c;

[0036] d is the immunohistochemical staining of the monoclonal antibody FMU-BAP31-No.1 in the liver cancer tissue of a patient with liver cancer III; d1 is a local magnified view of d;

[0037] e is the immunohistochemical staining of the monoclonal antibody FMU-BAP31-No.1 in the liver cancer tissue of a patient with liver cancer IV; e1 is a local magnified view of e.

[0038] Figure 4 Immunofluorescence staining of different liver cancer cell lines with the Cy5-BAP31 directly labeled monoclonal antibody prepared by the present invention. Blue represents the cell nucleus stained with DAPI, green represents BAP31, red represents SERPINE2, and yellow represents the result of co-localization of BAP31 and SERPINE2.

[0039] A is the immunofluorescence staining of BAP31, SERPINE2 and cell nuclei in Hep3b liver cancer cell line using Cy5-BAP31 direct-labeled monoclonal antibody and SERPINE2 rabbit polyclonal antibody;

[0040] B is the immunofluorescence staining of BAP31, SERPINE2 and cell nuclei in the MHCC97h liver cancer cell line using Cy5-BAP31 direct-labeled monoclonal antibody and SERPINE2 rabbit polyclonal antibody.

[0041] Figure 5 This figure shows the effect of the monoclonal antibody FMU-BAP31-No.1 prepared in the present invention on the scratch healing ability of Hep3b and MHCC97h liver cancer cell lines; in the figure, anti-BAP31 refers to the monoclonal antibody FMU-BAP31-No.1, and IgG isotype refers to unrelated IgG;

[0042] In the figure, A is a micrograph of cells in a scratch healing assay of Hep3b liver cancer cell line treated with monoclonal antibody FMU-BAP31-No.1 and irrelevant IgG. The first, second and third columns are micrographs of cells treated for 0 h, 24 h and 48 h, respectively. The first, second and third rows are micrographs of Hep3b liver cancer cells, Hep3b liver cancer cells treated with irrelevant IgG and Hep3b liver cancer cells treated with BAP31 antibody, respectively.

[0043] B is a micrograph of cells in a scratch healing assay of MHCC97h liver cancer cell line treated with monoclonal antibody FMU-BAP31-No.1 and irrelevant IgG. The first, second and third columns are micrographs of MHCC97h liver cancer cells, MHCC97h liver cancer cells treated with irrelevant IgG and MHCC97h liver cancer cells treated with BAP31 antibody, respectively.

[0044] C is a statistical graph showing the scratch healing ability of monoclonal antibody FMU-BAP31-No.1 and irrelevant IgG on Hep3b liver cancer cell line;

[0045] D is a statistical graph showing the scratch healing ability of monoclonal antibody FMU-BAP31-No.1 and irrelevant IgG on the MHCC97h original liver cancer cell line;

[0046] Figure 6 The effect of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention on the migration ability of Hep3b and MHCC97h liver cancer cell lines;

[0047] In the figure, A is a cell diagram showing the migration ability of Hep3b and MHCC97h liver cancer cell lines after treatment with the monoclonal antibody FMU-BAP31-No.1, where NC represents cells without any treatment, IgG isotype represents liver cancer cells treated with an irrelevant IgG antibody, and anti-BAP31 represents liver cancer cells treated with the monoclonal antibody FMU-BAP31-No.1; the first row represents Hep3b liver cancer cells, and the second row represents MHCC97h liver cancer cells;

[0048] B shows the effects of no treatment, IgG isotype treatment and anti-BAP31 treatment on the migration ability of Hep3b liver cancer cells;

[0049] C shows the effects of no treatment, IgG isotype treatment and anti-BAP31 treatment on the migration ability of MHCC97h liver cancer cells.

[0050] Figure 7 The effect of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention on subcutaneous tumor formation in nude mice of the Hep3b liver cancer cell line;

[0051] A shows the subcutaneous tumor formation of Hep3b liver cancer cell line in nude mice after treatment with anti-BAP31, IgG isotype and PBS;

[0052] B shows the effects of anti-BAP31, IgG isotype and PBS treatment on the weight of subcutaneous tumors in nude mice with Hep3b liver cancer cell line;

[0053] C shows the effects of anti-BAP31, IgG isotype and PBS treatment on the subcutaneous tumor volume of Hep3b liver cancer cell line in nude mice.

[0054] Figure 8 The effect of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention on the metastatic ability of MHCC97h cells in vivo;

[0055] In the figure, A is a CT scan image of nude mice treated with PBS after tail vein injection of MHCC97h liver cancer cells;

[0056] B is a CT scan image of nude mice treated with IgG isotype after tail vein injection of MHCC97h liver cancer cells;

[0057] C is a CT scan image of nude mice treated with anti-BAP31 after tail vein injection of MHCC97h liver cancer cells.

[0058] Figure 9The effect of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention on the survival period of nude mice bearing MHCC97h liver cancer cells;

[0059] A shows the effects of anti-BAP31 and PBS injection on the survival rate of nude mice bearing liver cancer cells at different days;

[0060] B shows the effect of anti-BAP31 and IgG isotype injection on the survival rate of nude mice bearing liver cancer cells at different days.

[0061] Figure 10 The effects of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention on the heart, liver, lung and kidney tissues of mice at therapeutic doses;

[0062] Figure A shows the effects of monoclonal antibody FMU-BAP31-No.1 treatment on the heart, liver, lung, and kidney tissues of mice;

[0063] B shows the effects of IgG isotype treatment on the heart, liver, lung, and kidney tissues of mice;

[0064] C shows the effects of PBS treatment on the heart, liver, lung, and kidney tissues of mice. DETAILED DESCRIPTION

[0065] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0066] The present invention immunized Balb / c mice with purified recombinant human BAP31 protein to produce a panel of mouse anti-BAP31 monoclonal antibodies. From these, a hybridoma cell line capable of stably secreting the high-affinity anti-BAP31 monoclonal antibody FMU-BAP31-No.1 was screened. Ascites fluid was then prepared to obtain the high-affinity anti-BAP31 monoclonal antibody FMU-BAP31-No.1. The uniqueness of the gene sequence and corresponding protein sequence, as well as their CDR sequences, were confirmed. This provides support for the development of a humanized genetically engineered anti-BAP31 antibody as a candidate tumor-targeting drug. The present invention is described in detail below, focusing on specific monoclonal antibody preparation methods, antibody activity testing, and sequence detection and uniqueness determination. This description is intended to explain, not to limit, the present invention.

[0067] Example 1: Preparation and identification of mouse anti-BAP31 high-affinity monoclonal antibody FMU-BAP31-No.1.

[0068] 1. Preparation of Monoclonal Antibody FMU-BAP31-No.1

[0069] 1. Experimental Materials

[0070] Balb / c mice were purchased from the Experimental Animal Center of the Fourth Military Medical University;

[0071] The preparation and purification of recombinant human BAP31 protein were based on the methods described in "Construction of truncated BAP31 / GST gene recombinant plasmid and expression, purification and identification of fusion protein".

[0072] The sequence of recombinant human BAP31 protein is:

[0073] SQQATLLASNEAFKKQAESASEAAKKYMEENDQLKKGAAVDGGKLDVGNAEVKLEEENRSLKADLQKLKDELASTKQKLEKAENQ.

[0074] Mouse myeloma cells SP2 / 0 were introduced from IMVS, Australia and preserved in our laboratory.

[0075] Normal Balb / c mouse peritoneal macrophages: Take a normal Balb / c mouse, open the peritoneal cavity on a sterile operating table, and repeatedly blow and wash the peritoneal cavity with 5 ml of 1640 culture medium, then finally aspirate it. At this time, the culture medium contains peritoneal macrophages.

[0076] 2. Preparation and purification of monoclonal antibodies

[0077] (1) Construction of hybridoma cell lines

[0078] Immunization: Balb / c mice were immunized with purified recombinant human BAP31 protein at a dose of 20 μg per mouse. Dilute the purified BAP31 protein with PBS to a 200 μg / ml solution. Mix 0.5 ml (100 μg) with 0.5 ml of adjuvant and grind to obtain 1 ml of the mixture. Each mouse was injected with 200 μl of the mixture at four sites, with a 50 μl injection volume at each site.

[0079] For the initial immunization, complete Freund's adjuvant (CFA) mixed with a protein solution in a 1:1 ratio was injected. Subsequent immunizations were administered with incomplete Freund's adjuvant (CFA) mixed with a protein solution in a 1:1 ratio, three weeks apart, at multiple subcutaneous injections for a total of four immunizations. Seven days after the final immunization, blood was collected for titer analysis to assess immune efficacy. Three weeks later, a booster immunization was performed with the same dose of antigen injected intraperitoneally (using protein alone). Three days later, the animals were sacrificed and spleens harvested for cell fusion.

[0080] Cell fusion: Logarithmically growing mouse myeloma SP2 / 0 cells were counted and an immune spleen cell suspension was prepared. After euthanizing mice, the peritoneal cavity was opened and the spleen removed under a sterile operating hood. The spleen was ground in a grinding mesh and added to 1640 medium to obtain a spleen cell suspension. The logarithmically growing myeloma SP2 / 0 cells and the immune spleen cell suspension were then mixed at a ratio of 1:10 and polyethylene glycol (PEG) was added for cell fusion to obtain a fused cell suspension. The fused cell suspension was plated into a 96-well plate containing feeder cells (normal Balb / c mouse peritoneal macrophages) and incubated at 37°C in a 5% CO2 incubator. After colonies emerged, positive clones were selected by indirect ELISA. Cells from wells containing positive clones were cloned using limiting dilution until a hybridoma cell line capable of stably secreting antibodies (continuously cultured in vitro for more than 6 months) was obtained. The secreted antibodies were then assayed for Ig subclass IgG1, Kappa light chain.

[0081] (2) Preparation of monoclonal antibody FMU-BAP31-No.1 and unrelated IgG

[0082] After obtaining a hybridoma cell line capable of stably secreting antibodies, the hybridoma cell line and SP2 / 0 cells were used to prepare ascites containing monoclonal antibodies and ascites containing irrelevant IgG, respectively, according to the mouse ascites preparation method. The specific method is as follows:

[0083] 1) Preparation of Bal-b / c mice: Purchase 20 Bal-b / c mice (which have a loose belly and typically produce more ascites after hybridoma cell injection) from the animal center of our university. Randomly divide the mice into two groups, ear-tagged each mouse, and inject 150 μL of liquid paraffin into each group. One week later, inject the hybridoma cells or SP2 / 0 cells.

[0084] 2) Intraperitoneal injection: The expanded hybridoma cell lines and SP2 / 0 cells were centrifuged, resuspended, and counted. The cell concentration of both groups was adjusted to 1×10 7 / mL, and 100 μL per mouse was intraperitoneally injected into two groups of Bal-b / c mice;

[0085] 3) Ascites Collection: After hybridoma cell injection, the mice were observed daily. A large amount of ascites was produced within 7-10 days, especially when the fur began to become rough, indicating the mice were on the verge of death and the amount of ascites was at its highest. The mice were anesthetized and sacrificed. The abdominal cavity was incised and the ascites were collected. The ascites were centrifuged at 12,000 rpm for 15 minutes at 4°C and the supernatant was collected. The supernatant of the ascites collected from each mouse was frozen at -80°C. After the ascites from all mice were collected, the antibody extraction was performed uniformly.

[0086] 4) Preparation of antibodies by saturated ammonium sulfate precipitation: Prepare saturated ammonium sulfate solution in advance, stir it in a water bath until crystals precipitate, and store it at room temperature until used. Remove the two sets of ascites produced by injection of hybridoma cells and SP2 / 0 from a -80°C freezer and melt on ice. Transfer the solution to a small beaker (ice bath), dilute with physiological saline, place the rotor on a magnetic stirrer, and add saturated ammonium sulfate solution dropwise to a final concentration of 45%. Stir on ice for 40 minutes, transfer the solution to a 50 mL centrifuge tube, and let it stand at 4°C overnight. Centrifuge the ascites precipitate that has stood overnight at 3000 rpm at 4°C for 20 minutes, resuspend the precipitate in the same volume of pre-chilled PBS as the ascites, aspirate it into an MD34 dialysis bag, clamp the ends, and dialyze it in 2 L of pre-chilled PBS at 4°C. Change the dialysate every 5-6 hours for a total of 5 changes.

[0087] 5) Concentrate the antibody and measure its concentration: After dialysis is complete, remove the dialysis bag, carefully remove the clamp, and use a pipette to aspirate the liquid in the bag into an ultrafiltration tube. Centrifuge at 4°C, 4000 rpm for 45 minutes to concentrate the antibody. After centrifugation, measure the concentration of the upper and lower liquids in the ultrafiltration tube. Aliquot and label the antibody and store it in a -80°C freezer. At this point, the ascites produced by the hybridoma cells will be used to prepare the BAP31 monoclonal antibody, while the ascites produced by the SP2 / 0 cells will be used to prepare the unrelated IgG for use as a control group.

[0088] II. Identification of Monoclonal Antibody FMU-BAP31-No.1

[0089] 1. Determination of the potency of the anti-BAP31 monoclonal antibody FMU-BAP31-No.1

[0090] The relative affinity of the monoclonal antibody before and after purification was determined using an indirect ELISA method. The coating antigen was recombinant human BAP31 protein, and the test samples were serially diluted ascites and purified monoclonal antibody. The detection antibody was a goat anti-mouse HRP-labeled antibody, and the substrate was ABTS. The high-affinity monoclonal antibody FMU-BAP31-No.1 was screened, with an ascites titer of 1×10 -7 After purification, the titer is 0.5 ng / mL, while the titer of ascites detected by indirect ELISA is generally 1×10 -5The above antibodies can be used.

[0091] 2. Western blot comparison of the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention and the commercial BAP31 rabbit polyclonal antibody

[0092] Commercial BAP31 rabbit polyclonal antibody was purchased from Proteintech with the catalog number 11200-1-AP.

[0093] Stable cells expressing or knocking down BAP31 (published in the paper, see PMID: 33363167) were lysed with RIPA buffer (containing protease inhibitors), and proteins were extracted and quantified by BCA assay. SDS-PAGE gels were prepared using an SDS-PAGE kit (YaZyme), and Western blots were performed. The primary antibodies used were the monoclonal antibody FMU-BAP31-No.1 (abbreviated as BAP31 mouse monoclonal antibody), a rabbit polyclonal antibody against BAP31, and a mouse monoclonal antibody against β-actin, at dilutions of 1:10,000, 1:2,500, and 1:5,000, respectively.

[0094] 3. Preparation of Cy5-BAP31 directly labeled monoclonal antibody (abbreviated as: Cy5-BAP31 antibody)

[0095] (1) The BAP31 mouse monoclonal antibody prepared by the present invention, i.e., the monoclonal antibody FMU-BAP31-No.1, was taken out from a -80°C freezer and thawed on ice;

[0096] (2) Cut the MD34 dialysis bag into about 10 cm long pieces with scissors, boil it in boiling water for 5 minutes, and then put it into ddH2O for later use;

[0097] (3) Prepare 2 L of 0.1 M NaHCO3 solution as the dialysate, adjust the pH to 8.3, and pre-cool in a 4°C refrigerator;

[0098] (4) Take 2 mL of melted BAP31 mouse monoclonal antibody and add it to the MD34 dialysis bag. Fold the two ends of the dialysis bag and clamp it with a clip to ensure that the liquid in the dialysis bag does not flow out.

[0099] (5) Place the dialysis bag containing BAP31 mouse monoclonal antibody in NaHCO3 dialysate and dialyze it in a 4°C refrigerator for 8 hours. This step is to ensure that the antibody is in an alkaline environment when it is linked to the fluorescent dye.

[0100] (6) After 8 hours, take out the BAP31 mouse monoclonal antibody. At this time, the antibody is in an alkaline environment. Add it to a small beaker and place it in the smallest rotor. Place the small beaker in crushed ice and place it on a magnetic stirrer. Slowly add 200 μL of 10 mg / mL Sulfo-Cy5-NHS fluorescent dye and stir in the dark for 45 minutes. Keep it on ice and avoid light during the whole process to obtain Cy5-labeled Cy5-BAP31 direct-labeled monoclonal antibody.

[0101] (7) The Cy5-BAP31 directly labeled monoclonal antibody was aspirated into the MD34 dialysis bag again, and 2L PBS was used as the dialysate. The dialysate was dialyzed at 4°C in the dark, and the dialysate was changed every 6 hours. The dialysate was dialyzed 6 to 8 times in total until the dialysate was no longer stained by the Cy5 dye. This step is to remove excess free Cy5 dye.

[0102] (8) Pack the dialyzed Cy5-BAP31 antibody in small packages, wrap it in tin foil and store it at -20°C until use.

[0103] 4. Flow cytometry detection of the binding of the BAP31 mouse monoclonal antibody (anti-BAP31) prepared by the present invention to the natural BAP31 molecules on the surface of liver cancer cells

[0104] Hep3b and MHCC97h liver cancer cells in the logarithmic growth phase were digested with EDTA-free trypsin, centrifuged, resuspended, washed twice with PBS, and counted. 1×10 6 Cells were added to the flow cytometry tubes, and each cell line was divided into two groups, one group as a negative control, and the other group as an experimental group for staining BAP31 molecules; the homemade Cy5-BAP31 antibody was taken out from the -80℃ refrigerator, and it was diluted step by step to 0.1μg / μL. 1μL of the diluted Cy5-BAP31 antibody was added to each flow cytometry tube of the experimental group, and placed in a 4℃ refrigerator and incubated in the dark for 30 minutes; after incubation, the cells were washed once with PBS, resuspended in 200μL PBS and then detected.

[0105] 5. Immunohistochemical staining

[0106] (1) Take out two human liver cancer tissue chips, LV2089 and LV1221 (purchased from Xi'an Aline Biotechnology Co., Ltd.), from the 4°C refrigerator and first bake them in an oven at 65°C-75°C for 1 hour;

[0107] (2) Dewaxing and hydration: Take the chip out of the oven and place it in the prepared solution tank in sequence: xylene for 15 minutes, xylene for the second time for 15 minutes, anhydrous ethanol for 10 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and finally immerse it in water;

[0108] (3) Antigen repair: Pour the prepared 1×EDTA antigen repair solution into the washing box, heat it in a microwave oven until it boils, then place the slide in it and continue heating on low heat for 20 minutes;

[0109] (4) Blocking: Take out the chip, wash with 1×TBST for 2 minutes, take out the slide and carefully dry it with absorbent paper, circle the tissue with an immunohistochemistry pen, add 5% BSA blocking solution, and let it stand for 15 minutes;

[0110] (5) Primary antibody incubation: dilute the homemade BAP31 mouse monoclonal antibody (anti-BAP31) with blocking solution at a dilution ratio of 1:100, drop it onto the chip to cover the tissue, place it in a humidified chamber, and incubate at room temperature for 1 h.

[0111] (6) Secondary antibody incubation: Place the chip in 1×TBST and wash for 5 minutes. After changing the solution three times, add Dako Real universal immunohistochemistry secondary antibody and incubate at room temperature for 40 minutes.

[0112] (7) Color development: Place the chip in 1×TBST and wash for 5 min. Change the solution three times and then add DAB-H2O2 for color development for 1 min. Rinse with distilled water.

[0113] (8) Restain the nucleus: Restain with hematoxylin for 5 minutes, wash the slide, and observe the staining under a microscope. If the staining is excessive, use hydrochloric acid alcohol differentiation;

[0114] (9) Dehydration: Dehydrate the sample in the following solutions: 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, anhydrous ethanol for 5 min, and xylene for 10 min.

[0115] (10) Seal the slide: Seal the slide with gum and observe it under a microscope after it is completely dry.

[0116] 6. Immunofluorescence (IF) staining

[0117] (1) Take Hep3b and MHCC97h liver cancer cells in the logarithmic growth phase, digest, centrifuge, resuspend, count, and adjust the cell concentration to 1×10 5 Cells / mL were used to plate confocal microplates. The bottom of each microplate was moistened with culture medium in advance, and then 1.5 mL of the cell suspension with the adjusted concentration was added. The cells were then cultured overnight in a cell culture incubator at 37°C and 5% CO2.

[0118] (2) The next day, place ten small dishes under a microscope to ensure that the cell density is approximately 30% to 40%, which is suitable for IF staining. Pour out all the culture medium in the small dishes and gently wash each dish with 2 mL of PBS.

[0119] (3) After discarding the PBS, add 1 mL of cold acetone and then 1 mL of paraformaldehyde to each dish and fix them at room temperature for 10 minutes. After multiple experiments, it was found that this combination can better fix the cells;

[0120] (4) Aspirate the fixative, gently wash once with PBS, then add 1-2 mL of Triton X-100 to each dish for cell perforation and leave at room temperature for 10 minutes.

[0121] (5) Aspirate the punching solution, gently wash once with PBS, then add 1-2 mL of 5% BSA blocking solution to each dish and let it stand at room temperature for 10 minutes;

[0122] (6) Dilute the primary antibody (anti-SERPINE2 rabbit polyclonal antibody (Proteintech, 11303-1-AP)) with the above blocking solution at a dilution ratio of 1:50 for use.

[0123] (7) Aspirate the blocking solution in the small dish, wipe the edge of the liquid with a cotton swab, add 60 μL of the diluted primary antibody to each depression of the small dish, and incubate at room temperature for 2 h;

[0124] (8) Aspirate the primary antibody solution and wash with 1×TBST buffer, adding 2 mL each time and placing on a shaker for 5 minutes, for a total of three washes;

[0125] (9) Dilute Cy3-donkey anti-rabbit secondary antibody and homemade Cy5-BAP31 directly labeled monoclonal antibody at 1:50, mix well, add 60 μL to each depression of the small dish, and incubate at room temperature for 1 h;

[0126] (10) Aspirate and discard the secondary antibody and directly labeled monoclonal antibody solution, wash with 1×TBST buffer, add 2 mL each time and place on a shaker to wash for 5 minutes, for a total of three washes;

[0127] (11) Add one drop of ready-to-use DAPI to the depression of the small dish, and incubate at room temperature for 10 minutes;

[0128] (12) Aspirate the DAPI solution and wash with 1×TBST buffer, adding 2 mL each time and placing on a shaker for 5 minutes, for a total of three washes;

[0129] (13) Finally, add 1 mL of PBS to each small dish, protect from light, and store in a refrigerator at 4°C. Use it for laser confocal microscopy observation as soon as possible.

[0130] 3. Identification results of monoclonal antibody FMU-BAP31-No.1

[0131] 1. Determination of the potency of the BAP31 monoclonal antibody FMU-BAP31-No.1

[0132] The results showed that the titer of BAP31 monoclonal antibody FMU-BAP31-No.1 in ascites was 1×10 -7 The titer after purification was 0.5 ng / mL.

[0133] 2. Western blot comparison of monoclonal antibody FMU-BAP31-No.1 and commercial BAP31 rabbit polyclonal antibody

[0134] To verify the effectiveness of the homemade BAP31 antibody, we used it together with the commercial BAP31 rabbit polyclonal antibody as Western blot experimental antibodies to compare their effects. We extracted proteins from Hep3b stably transfected cell lines with stable upregulation, knockdown of BAP31 and control, and detected their BAP31 expression levels. The results are shown in Figure 2. Figure 1 As shown, the homemade BAP31 antibody can well recognize the BAP31 molecule and achieve the expected effect.

[0135] 3. Flow cytometry detection of the binding of the BAP31 mouse monoclonal antibody (anti-BAP31) prepared by the present invention to the natural BAP31 molecules on the surface of liver cancer cells

[0136] In addition, we also used the homemade Cy5-labeled Cy5-BAP31 direct-labeled monoclonal antibody as a flow cytometry antibody to see if it can bind to the BAP31 antibody on the cell membrane surface. We used Hep3b and MHCC97h original cell lines for flow cytometry experiments. Each cell line was divided into two groups, a negative control group and an experimental group with Cy5-BAP31 direct-labeled monoclonal antibody. After observation on the flow cytometer, the results were as follows: Figure 2 As shown, it can be seen that the Cy5-BAP31 antibody can bind well to the BAP31 molecules on the cell membrane surface.

[0137] 4. Immunohistochemical staining

[0138] The results are as follows Figure 3 As shown in the results, BAP31 is abnormally highly expressed in HCC tissues, and its expression level tends to increase with clinical stage.

[0139] 5. Immunofluorescence (IF) staining

[0140] The results are as follows Figure 4 As shown in the figure, we used homemade Cy5-BAP31 direct-labeled antibody, SERPINE2 rabbit polyclonal antibody and DAPI to stain BAP31, SERPINE2 and cell nuclei of Hep3b and MHCC97h original cell lines in order to observe the co-localization of BAP31 and SERPINE2 in cells. Figure 4As shown, blue represents the cell nucleus stained with DAPI, green represents BAP31, red represents SERPINE2, and yellow is the result of co-localization of BAP31 and SERPINE2. It can be observed that BAP31 can co-localize with SERPINE2 in Hep3b and MHCC97h original cell lines.

[0141] Example 2: The monoclonal antibody FMU-BAP31-No.1 prepared in Example 1 was used in intervention experiments on liver cancer cells in vivo and in vitro.

[0142] 1. Experimental Methods

[0143] 1. Cell scratch test

[0144] (1) Take Hep3b and MHCC97h original liver cancer cells in the logarithmic phase, digest, centrifuge, resuspend, count, and plate 2 mL per well in a 6-well plate. Be careful to keep the 6-well plate at a 30° angle to the operating table during inoculation until it is placed flat in the cell culture incubator without shaking to ensure uniform platooning of the 6-well plate.

[0145] (2) Culture at 37°C and 5% CO2 until the density is approximately 80%. Use a sterilized 200 μL pipette tip to draw three straight lines along the cover of each well of the 6-well plate. Gently rinse the floating cells with PBS, replace the culture medium with a medium containing 2% FBS, and divide the cells into three groups: a blank group, an irrelevant IgG group, and a BAP31 mouse monoclonal antibody (anti-BAP31) group. Add 5 μL each of irrelevant IgG and the BAP31 mouse monoclonal antibody (anti-BAP31) prepared in Example 1 to the cell culture medium (final concentration 100 μg / mL). Place the 6-well plate under a microscope and take a picture. This time is recorded as 0 h.

[0146] (3) After 24 hours and 48 hours, the 6-well plates were placed under a microscope and photographed. The photos were saved and analyzed to analyze the differences in the healing speed of the scratches among different groups.

[0147] 2. Cell migration assay

[0148] (1) Take Hep3b and MHCC97h original liver cancer cells in the logarithmic phase, replace the culture medium with serum-free medium and culture for 4 hours, then digest, centrifuge, resuspend in serum-free medium, count, and adjust the cell concentration to 3×10 5 Pieces / mL for standby use;

[0149] (2) Take a 24-well plate and add 600 μL of DMEM medium containing 20% ​​FBS to each well. Then, place a transwell chamber in the well and add 100 μL of serum-free medium to the chamber. The cells are divided into three groups: a blank group, an irrelevant IgG group, and a BAP31 mouse monoclonal antibody (anti-BAP31) group. Add 2 μL of irrelevant IgG and 2 μL of BAP31 mouse monoclonal antibody (anti-BAP31) to the upper chamber, respectively. Be careful not to have bubbles on the lower side of the chamber.

[0150] (3) Mix the prepared cell suspension again and inoculate 100 μL per well into the transwell chamber, keeping the liquid levels inside and outside the chamber level, and incubate at 37°C and 5% CO2 for 48 h.

[0151] (4) After 48 hours, take out the transwell chamber, rinse it gently with PBS twice, and carefully wipe off the cells inside the chamber with a cotton swab; then place it in pre-cooled anhydrous ethanol for 10 minutes;

[0152] (5) Take the chamber out of the anhydrous ethanol and place it in the crystal violet staining solution. Stain at room temperature for 10 minutes.

[0153] (6) Recover the crystal violet staining solution and rinse with distilled water until the chamber is clean. Observe under a microscope and take photos to record.

[0154] 3. Nude mouse subcutaneous tumor intervention experiment

[0155] (1) Balb / c nude mice were purchased from the Experimental Animal Center of the Fourth Military Medical University according to the experimental requirements. In this experiment, three groups of nude mice were used to observe the effect of BAP31 mouse monoclonal antibody on subcutaneous tumor formation, with 5 mice in each group. The newly purchased nude mice were kept in the animal center for one week to adapt to the environment.

[0156] (2) The Hep3b original cell line was expanded and cultured, and 2×10 6 Calculate the number of cells required for the experiment;

[0157] (3) All cells in the expanded culture were digested, centrifuged, resuspended in PBS, washed, and finally resuspended in PBS to a cell concentration of 2×10 7 / mL;

[0158] (4) Bag the prepared cell suspension, 1 mL syringe, cotton swab, alcohol spray bottle, mask, gloves and other items and take them to the animal center for the experiment;

[0159] (5) Take out the nude mouse on the sterile operating table, disinfect the skin on the lower right back of the nude mouse to be injected, mix the cell suspension again, aspirate the cell suspension with a syringe and remove the bubbles, and inject 100 μL subcutaneously into each nude mouse;

[0160] (6) All nude mice were randomly divided into three groups, including two control groups (PBS group and irrelevant IgG group) and one experimental group (BAP31 mouse monoclonal antibody). Starting from the fourth day after subcutaneous injection of liver cancer cells, each mouse was intraperitoneally injected with BAP31 mouse monoclonal antibody or irrelevant IgG at a dose of 10 mg / kg. The PBS injection volume was the same as the BAP31 mouse monoclonal antibody injection volume. The treatment was repeated twice a week. The length, width and height of the tumor were measured and recorded every three days.

[0161] (7) After three weeks of treatment, the mice were killed, the tumors were removed, photographed, and weighed. In addition, the heart, liver, lung, and kidney tissues were taken and fixed together with the tumor tissue in 4% paraformaldehyde, embedded in paraffin, and sectioned for immunohistochemical staining.

[0162] 4. Nude mouse tail vein transfer experiment

[0163] (1) Nude mice were purchased from the animal center according to the experimental needs. In this experiment, three groups of nude mice were used to observe the intervention of anti-BAP31 monoclonal antibody on tail vein lung metastasis, with 10 mice in each group. The newly purchased nude mice were kept in the animal center for one week to adapt to the environment.

[0164] (2) Expand the culture of MHCC97h original cell line and inject 2×10 6 Calculate the number of cells required for the experiment;

[0165] (3) All cells in the expanded culture were digested, centrifuged, resuspended in PBS, washed, and finally resuspended in PBS to a cell concentration of 2×10 7 / mL;

[0166] (4) Bag the prepared cell suspension, 1 mL syringe, cotton swab, alcohol spray bottle, mask, gloves and other items and take them to the animal center for the experiment;

[0167] (5) Remove the nude mouse from the sterile operating table, fix its body and expose the tail vein, mix the cell suspension again, aspirate the cell suspension with a syringe and remove the bubbles, and inject 100 μL into the tail vein of each nude mouse;

[0168] (6) All nude mice were randomly divided into three groups: two control groups (PBS group and irrelevant IgG group) and one experimental group (BAP31 mouse monoclonal antibody group). Starting from the 7th day after subcutaneous injection of liver cancer cells, each mouse was intraperitoneally injected with anti-BAP31 monoclonal antibody or irrelevant IgG at a dose of 10 mg / kg. The PBS injection volume was the same as the BAP31 mouse monoclonal antibody injection volume, and the treatment was repeated twice a week.

[0169] (7) After six weeks of treatment, the mice were anesthetized with isoflurane and scanned under a small animal Micro-CT machine to check the tumor metastasis in the body; the remaining nude mice were kept until death, and the death time of each nude mouse was recorded.

[0170] 2. Experimental Results

[0171] 1. Cell scratch test

[0172] The results are as follows Figure 5 As shown above, the experimental results show that the BAP31 mouse monoclonal antibody prepared by the present invention can bind to the BAP31 molecules on the membrane surface. Therefore, we used BAP31 mouse monoclonal antibody to co-culture with cells to see whether it can affect the biological behavior of liver cancer cells.

[0173] We used Hep3b and MHCC97h liver cancer cell lines to perform a cell wound healing experiment. The two cell lines were divided into three groups: one as a blank group, one as an isotype IgG group (IgG ositype), and the other as a BAP31 antibody experimental group (anti-BAP31). Isotype IgG and BAP31 antibodies were added at 0h and the medium was not changed until 48h. The results are shown in Figure 2. Figure 5 As shown in the figure, compared with the blank group and isotype IgG group, BAP31 antibody significantly inhibited the scratch healing ability of liver cancer cells, and the difference was statistically significant.

[0174] 2. Cell migration assay

[0175] The results are as follows Figure 6 As shown, in addition, we also used Hep3b and MHCC97h liver cancer cell lines to perform transwell migration experiments, and the cells were divided into three groups: one group as a blank group, one group as an isotype IgG group, and the other group as a BAP31 antibody experimental group. At 0h, isotype IgG and BAP31 antibodies were added to the upper chamber of the transwell and the medium was not changed until 48h. Figure 6 As shown in Figure 2, compared with the blank group and the isotype IgG group, BAP31 antibody significantly inhibited the migration ability of liver cancer cells, and the difference was statistically significant.

[0176] 3. Nude mouse subcutaneous tumor intervention experiment

[0177] The results are as follows Figure 7As shown in the figure, nude mice were subcutaneously injected with Hep3b liver cancer cells. Four days after injection, BAP31 antibody intervention was initiated. Three groups of nude mice were treated with BAP31 antibody, isotype IgG, and PBS, respectively, at a dose of 10 mg / kg twice weekly. After three weeks of treatment, tumors were removed, photographed, and weighed. Starting on the seventh day after injection, the length, width, and height of the tumors were measured and recorded every three days. Compared with the PBS and isotype IgG groups, the nude mice treated with BAP31 antibody had slower tumor growth, smaller tumor volume, and lighter tumor weight, with statistically significant differences. These results demonstrate that BAP31 antibody significantly inhibits the subcutaneous tumorigenesis of liver cancer cells.

[0178] The results are as follows Figure 8 To further explore the effect of BAP31 on the in vivo migration of liver cancer cells, we injected nude mice with MHCC97h liver cancer cells via the tail vein. Starting one week after injection, we administered BAP31 antibodies. Three groups of nude mice were treated with BAP31 antibodies, isotype IgG, and PBS, respectively, at a dose of 10 mg / kg twice weekly. After six weeks of treatment, small animal micro-CT scans were performed to observe tumor metastasis. Mice in the PBS and isotype IgG groups developed metastatic foci in the liver or lungs, while mice in the BAP31 antibody-treated group showed almost no metastatic foci.

[0179] The results are as follows Figure 9 As shown, we also recorded the survival time of each nude mouse. After summarizing all the time, we found that compared with the PBS group and the isotype IgG group, the BAP31 antibody significantly prolonged the survival of nude mice injected with liver cancer cells, and the difference was statistically significant.

[0180] The results are as follows Figure 10 To investigate the effects of BAP31 monoclonal antibody treatment on the heart, liver, lung, and kidney of nude mice, we removed the hearts, livers, lungs, and kidneys from the PBS, isotype IgG, and BAP31-treated groups, embedded them in paraffin, and then sectioned and stained them with HE. Compared with the PBS and isotype IgG groups, the heart, liver, lung, and kidney tissues of the BAP31-treated mice were no different. This result suggests that at the therapeutic dose in this experiment, BAP31 monoclonal antibody had no effect on the heart, liver, lung, and kidney tissues of mice.

[0181] Example 3:

[0182] 1. Cloning of the heavy chain variable region gene of the anti-BAP31 monoclonal antibody FMU-BAP31-No.1

[0183] (1) Hybridoma cells secreting the monoclonal antibody FMU-BAP31-No.1 were cultured according to the above steps and cultured in RPMI 1640 medium containing 10% calf serum in an incubator at 37°C and 5% CO2 until the logarithmic growth phase.

[0184] (2) Extraction of total RNA and synthesis of the first strand of cDNA

[0185] Total RNA from hybridoma cells in the logarithmic growth phase was extracted using an RNA extraction kit (Qingke Biotechnology), and the specific operation steps were carried out according to the manufacturer's instructions. A cDNA first-strand synthesis kit was purchased from Nanjing Novozymes Biotechnology, and after obtaining total RNA, reverse transcription was performed to synthesize the first-strand cDNA according to the manufacturer's instructions.

[0186] (3) RT-PCR amplification of the heavy chain variable region and light chain variable region gene sequences of the monoclonal antibody FMU-BAP31-No.1

[0187] One-step RT-PCR amplification kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd., and the VH and VL genes of the monoclonal antibody FMU-BAP31-No.1 were amplified according to the kit instructions;

[0188] RT-PCR was performed using VH-F as the upstream primer and VH-B as the downstream primer, and total RNA of hybridoma cells as the template to amplify the heavy chain variable region gene sequence;

[0189] RT-PCR was performed using VL-F as the upstream primer and VL-B as the downstream primer, and total RNA of hybridoma cells as the template to amplify the light chain variable region gene sequence;

[0190] VH-F: tgaggagacggtgaccgtggtcccttggccccag; VH-B: aggtsmarctgcagsagtcwgg.

[0191] VL-F: gttagatctccagcttggtccc; VL-B: gacattcagctgacccagtctcca.

[0192] (4) Cloning and screening of PCR amplification products

[0193] The PCR product was subjected to 1.5% agarose gel electrophoresis, and the PCR amplified fragment was recovered using a mini-gel recovery kit (purchased from Omega, USA). This fragment was inserted into the pMD-T18 vector (purchased from TakaRa) using a DNA ligation kit (purchased from TakaRa) according to the manufacturer's instructions, using an A-tail. The ligation product was transformed into E. coli (purchased from China General Microorganism Culture Collection, CGMCC, Beijing) and inoculated into Amp-resistant LB agar culture plates and cultured overnight at 37°C.

[0194] Select colonies from LB agar culture plates and shake in Amp-resistant LB medium at 37°C overnight. Using 1 μL of the bacterial solution as a template, screen for recombinant E. coli clones by PCR using primers designed for the heavy chain variable region.

[0195] The obtained recombinant-positive E. coli clones were cultured and the bacterial suspension was sent to Shanghai Bioengineering Technology Service Co., Ltd. for gene sequencing. The gene sequence of the heavy chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence encoded by it is shown in SEQ ID NO. 3. The gene sequence of the light chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence encoded by it is shown in SEQ ID NO. 4.

[0196] The variable region structure was analyzed using IMGT / V-QUEST, and it was determined that the heavy chain variable region contained three CDR regions, CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 was shown in SEQ ID NO.5; the amino acid sequence of CDR2 was shown in SEQ ID NO.6; and the amino acid sequence of CDR3 was shown in SEQ ID NO.7.

[0197] SEQ ID NO.5: Gly-Tyr-Thr-Phe-Thr-Ser-Tyr-Tyr;

[0198] SEQ ID NO.6: Ile-Tyr-Pro-Gly-Asn-Val-Asn-Thr;

[0199] SEQ ID NO. 7: Ala-Arg-Asn-Trp-Ala-Tyr.

[0200] It should be noted that the anti-tumor antigen BAP31 monoclonal antibody FMU-BAP31-No.1, BAP31 mouse monoclonal antibody, and anti-BAP31 represent the same substance; Cy5-BAP31 directly labeled monoclonal antibody, Cy5-BAP31 antibody, and Cy5-BAP31 all represent the same meaning. SERPINE2 rabbit polyclonal antibody and SERPINE2 are synonymous. Isotype IgG, IgG, IgG isotype, and unrelated IgG all represent the same meaning. Experiments have demonstrated that the monoclonal antibody FMU-BAP31-No.1 prepared by the present invention can be used for Western blotting, flow cytometry, immunohistochemistry, and immunofluorescence staining to detect the BAP31 antigen. In vitro and in vivo experiments have confirmed that the antibody can inhibit the wound healing, migration, subcutaneous tumor formation, and metastasis of liver cancer cells. The uniqueness of its heavy chain gene sequence and corresponding protein sequence, as well as its CDR sequence, have been confirmed. This provides support for the construction of various anti-BAP31 genetically engineered antibodies for therapeutic, detection, and preventive applications in the medical and life science fields.

[0201] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0202] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An anti-tumor antigen BAP31 monoclonal antibody, characterized in that: The monoclonal antibody is FMU-BAP31-No.1, which includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the monoclonal antibody FMU-BAP31-No.1 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the monoclonal antibody FMU-BAP31-No.1 is shown in SEQ ID NO.

4.

2. The anti-tumor antigen BAP31 monoclonal antibody according to claim 1, characterized in that The heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.5; the amino acid sequence of CDR2 is shown in SEQ ID NO.6; and the amino acid sequence of CDR3 is shown in SEQ ID NO.

7.

3. A Cy5-BAP31 directly labeled monoclonal antibody, characterized in that: Obtained by labeling the monoclonal antibody according to claim 1 or 2 with Cy5.

4. Use of the Cy5-BAP31 directly labeled monoclonal antibody according to claim 3 in the preparation of a BAP31 detection reagent.

5. Use of the anti-tumor antigen BAP31 monoclonal antibody according to any one of claims 1 to 2 in the preparation of an immunoassay tool for detecting BAP31.

6. Use of the anti-tumor antigen BAP31 monoclonal antibody according to claim 5 in the preparation of an immunoassay tool for detecting BAP31, characterized in that: The immunoassay tool is a kit, a chip or a test paper.

7. Use of the anti-tumor antigen BAP31 monoclonal antibody according to claim 6 in the preparation of an immunoassay tool for detecting BAP31, characterized in that: The kit is an immunohistochemistry detection kit.

8. Use of the anti-tumor antigen BAP31 monoclonal antibody according to any one of claims 1 to 2 in the preparation of a preparation for treating liver cancer.

9. Use of the anti-tumor antigen BAP31 monoclonal antibody according to claim 8 in the preparation of a preparation for treating liver cancer, characterized in that: The preparation is a medicine; the medicine has the following effects: Inhibit the proliferation of liver cancer cells; Inhibits the migration ability of liver cancer cell lines; Inhibits subcutaneous tumor formation of liver cancer cell lines in nude mice; Inhibit the metastatic ability of liver cancer cells in vivo; Improve the survival of nude mice.

10. Use of the anti-tumor antigen BAP31 monoclonal antibody according to claim 9 in the preparation of a preparation for treating liver cancer, characterized in that: The liver cancer cells are Hep3b or MHCC97h.

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