Anti-human BCOR protein monoclonal antibody and hybridoma cell strain and application thereof

Abstract

The invention relates to the technical field of immune globulin, in particular to an anti-human BCOR protein monoclonal antibody and a hybridoma cell strain and application thereof. The invention provides an anti-human BCOR protein monoclonal antibody which is secreted by a hybridoma cell strain OTI3D11, the hybridoma cell strain OTI3D11 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 28, 2024, and the preservation number of the hybridoma cell strain OTI3D11 is CGMCC No.46244. The anti-human BCOR protein monoclonal antibody can be used for preparing anti-human BCOR protein. The anti-human BCOR protein monoclonal antibody secreted by the hybridoma cell strain OTI3D11 disclosed by the invention has very high specificity and sensitivity. Therefore, the anti-BCOR monoclonal antibody secreted by the hybridoma cell strain OTI3D11 can be used for immunohistochemical detection so as to detect the expression condition of related tumor BCOR, and a basis is provided for diagnosis of patients by clinical doctors.

Description

Technical Field

[0001] The present application relates to the technical field of immunoglobulins, and in particular to anti-human BCOR protein monoclonal antibodies and hybridoma cell lines and applications thereof. Background Art

[0002] The BCOR (BCL6 co-repressor) gene is located on chromosome Xp11.4 and is a POZ / zinc finger transcriptional repressor consisting of 1755 amino acids. The function of the BCOR protein is related to its two main domains: the BCL6 binding domain binds to BCL6 to jointly inhibit gene transcription and play an anti-apoptotic role; the PUFD domain binds to the polycomb repressive complex PRC1, inhibits the ubiquitination modification of histone H2A, and inhibits gene transcription through epigenetic mechanisms. It plays an important role in normal early embryonic development and is expressed in a variety of tumors. The changes in the BCOR gene in tumors mainly include BCOR rearrangement and BCOR internal tandem duplication. It has been found that the partner genes that rearrange with BCOR include CCNB3, MAML3, ZC3H7B and many others. Tumors with abnormal BCOR gene include BCOR-altered sarcoma, BCOR-CCNB3 sarcoma, BCOR-ITD sarcoma, renal clear cell sarcoma, high-grade endometrial stromal sarcoma, central nervous system tumors (including high-grade CNS-PNET with BCOR abnormalities, medulloblastoma, retinoblastoma, glioma, etc.), blood system-related tumors (such as MDS in myeloid tumors, MDS with multiple lineage abnormalities, MDS with increased primitive cells, AML with trisomy 11, AML with trisomy 13, etc.), etc.

[0003] The expression of BCOR has differential diagnostic value in a variety of tumors. For example, BCOR is a specific marker for high-grade endometrial stromal sarcoma (HGESS) with YWHAE-NUTM2 fusion. The expression of BCOR is also helpful in diagnosing small round blue cell sarcoma with BCOR gene abnormalities. In renal clear cell sarcoma, BCOR positive expression is part of its characteristic histomorphology. BCOR is mainly localized to the cell nucleus in immunohistochemical testing. In BCOR-CCNB3 sarcoma, BCOR shows nuclear positivity, often with strong and diffuse nuclear labeling. This nuclear positive expression helps distinguish BCOR-related tumors from other types of tumors.

[0004] Therefore, it is crucial to develop highly specific and sensitive monoclonal antibodies that recognize BCOR protein for differential diagnosis of related tumors. Summary of the invention

[0005] In view of this, the present application provides an anti-human BCOR protein monoclonal antibody, a hybridoma cell line capable of secreting the anti-human BCOR protein monoclonal antibody, and applications of the anti-human BCOR protein monoclonal antibody and hybridoma cell line.

[0006] On the one hand, the present application provides a monoclonal antibody or an antigen-binding fragment against human BCOR protein, wherein the monoclonal antibody or the antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the amino acid sequence of LCDR1 comprises QSLFNSRTRKNY (SEQ ID NO.4), the amino acid sequence of LCDR2 comprises DTS (SEQ ID NO.5), and the amino acid sequence of LCDR3 comprises KQSY (SEQ ID NO.6).

[0007] Furthermore, the heavy chain variable region comprises HCDR1-3, the amino acid sequence of the HCDR1 comprises GFSIKDTY (SEQ ID NO.8), the amino acid sequence of the HCDR2 comprises IDPANGDT (SEQ ID NO.9), and the amino acid sequence of the HCDR3 comprises DYRGEWFVY (SEQ ID NO.10).

[0008] Further, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.3, preferably, an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0009] Furthermore, the light chain variable region has a total length of 109 amino acids, the four domains of its FR have amino acids 26, 17, 36 and 11 respectively, the three domains of LCDR1-3 have amino acids 12, 3 and 4 respectively, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-38aa, 56aa-58aa and 95aa-98aa respectively.

[0010] Further, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.7, preferably, an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0011] Furthermore, the total length of the heavy chain variable region is 113 amino acids, the amino acid numbers of the four domains of its FR are 24, 17, 36 and 11 respectively, the amino acid numbers of the three domains of HCDR1-3 are 8, 8 and 9 respectively, and the HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-102aa respectively.

[0012] On the other hand, the present application also provides a hybridoma cell line capable of secreting anti-human BCOR protein monoclonal antibodies or antigen-binding fragments, the hybridoma cell line being a mouse anti-human BCOR monoclonal hybridoma cell line OTI3D11, whose deposit number is CGMCC No.46244, and which was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on November 28, 2024. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] On the other hand, the present application also provides a monoclonal antibody or antigen-binding fragment against human BCOR protein, wherein the monoclonal antibody or antigen-binding fragment against human BCOR protein is obtained by secretion of the hybridoma cell line OTI3D11.

[0014] On the other hand, the present application also provides an application of the monoclonal antibody or antigen-binding fragment, characterized in that the application is any of the following: 1) labeling of human BCOR protein in cells or tissues; 2) preparing an immunohistochemical detection kit for human BCOR protein.

[0015] Furthermore, the cells are cancer cells or normal tissue cells, preferably endometrial stromal sarcoma tissue.

[0016] On the other hand, the present application also provides an immunohistochemical detection kit for human BCOR protein, wherein the immunohistochemical detection kit comprises an antibody detection reagent, and the antibody detection reagent contains the monoclonal antibody or antigen fragment.

[0017] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color developing solution, and hematoxylin staining solution.

[0018] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultra-sensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution; preferably, the secondary antibody reagent is an ultra-sensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0019] Preservation Information Biomaterial: OTI3D11 Classification and naming: Mouse anti-human BCOR monoclonal hybridoma cell line Deposit number: CGMCC No.46244 Deposit date: November 28, 2024 Depository: China General Microbiology Center (CGMCC) Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] Compared with the prior art, the anti-human BCOR protein monoclonal antibody or antigen-binding fragment provided by the present application can specifically bind to the human BCOR protein, significantly improving the specificity, accuracy and reliability of BCOR protein immunoassay; at the same time, the hybridoma cell line OTI3D11 provided can also stably secrete anti-human BCOR protein monoclonal antibodies. Secondly, using the anti-human BCOR protein monoclonal antibody and kit of the present application, the IHC method is used to detect tissues such as endometrial stromal sarcoma, and the BCOR protein expressed in the cells can be clearly detected, indicating that the anti-human BCOR protein monoclonal antibody of the present application can be well used for immunohistochemical detection of BCOR protein in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation of the present application, the drawings required for describing the specific implementation are briefly introduced below.

[0022] Figure 1 This is a graph showing the results of Western blot detection of the recombinant BCOR protein in Example 2, in which the expression of the recombinant BCOR protein in E. coli cells was detected using anti-HIS antibody, wherein lane L is the detection result using the lysate of E. coli cells transfected with an empty vector as the antigen, and lane R is the detection result using the lysate of E. coli cells transfected with a pET23a-rBCOR plasmid as the antigen.

[0023] Figure 2 This is the result of SDS-PAGE of BCOR protein in Example 2. The recombinant BCOR protein was purified by nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining.

[0024] Figure 3 This is a schematic diagram of the results of IHC detection of endometrial stromal sarcoma using the monoclonal antibody secreted by the hybridoma cell line OTI3D11 in Example 6. DETAILED DESCRIPTION

[0025] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals, or conditions recommended by manufacturers.

[0026] Example 1 Construction of BCOR protein recombinant expression plasmid The BCOR gene NM_001123385 was selected from Genebank. According to the amino acid sequence characteristics of BCOR, the 1225-1465 amino acid fragment of BCOR was designed as an immunogen (the corresponding nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2). Primers were designed, restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene, respectively, and the gene was inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rBCOR.

[0027] Example 2 Expression and purification of recombinant BCOR protein (1) Transformation of E. coli cells: Thaw 100 μl competent cells on ice, add recombinant plasmid DNA and mix gently. Heat shock at 42°C for 90 seconds after ice bath for 30 minutes, and then continue to ice bath for 1-2 minutes. Add 500 μl fresh antibiotic-free LB medium in a clean bench, incubate at 37°C on a shaker for 45 minutes, take an appropriate amount of bacterial solution and evenly spread it on the plate containing antibiotics, and invert the culture dish in a 37°C constant temperature incubator for overnight culture.

[0028] (2) Lysis of cells: Pick a single clone and place it in fresh culture medium. Cultivate at 37°C and 200 rpm until the OD value reaches 0.4-0.6. Add IPTG (final concentration 1 mM) and induce the culture for 7 hours. Collect the cells by centrifugation, then resuspend the cells in lysis buffer, ultrasonically disrupt for 20 minutes, and centrifuge at 12000 rpm at 4°C for 20 minutes to collect the supernatant. Take a small amount of supernatant protein and use anti-His antibody for WB identification, see Figure 1 .

[0029] Depend on Figure 1 The results showed that the E. coli cell lysate transfected with pET23a-rBCOR plasmid was located in lane R, with an obvious specific band at 31kD, and the molecular weight was consistent with the theoretical molecular weight of the selected amino acid fragment, while there was no band of corresponding size in lane 1 of the control lysate transformed with an empty vector, indicating that the cells specifically expressed the recombinant BCOR protein.

[0030] (3) Nickel affinity chromatography column purification: The nickel column was equilibrated with buffer, the supernatant was filtered through a 0.45 μm filter membrane, and then the sample was loaded and the outflow was collected. Unbound proteins were removed by elution with buffer, and finally eluted with eluents containing different concentrations of imidazole. After collection, the eluted proteins that met the requirements were combined and 10% glycerol was added. The purified recombinant BCOR protein was identified by SDS-PAGE electrophoresis, see Figure 2 .

[0031] Depend on Figure 2 The results showed that the purified protein had an obvious specific band at 31kD in the SDS-PAGE electrophoresis gel image, which was consistent with the theoretical predicted molecular weight of the immunogenic amino acid fragment, indicating that a recombinant BCOR protein with good purity had been obtained.

[0032] Example 3 Preparation and screening of monoclonal antibodies secreted by OTI3D11 against human BCOR protein BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized with purified recombinant BCOR protein (hereinafter referred to as BCOR antigen) according to standard methods. The specific method is as follows: (1) Animal immunization: The purified BCOR antigen was emulsified with complete Freund's adjuvant and immunized into 6-8 week old BALB / c mice by subcutaneous or intraperitoneal injection at a dose of 60 μg / mouse. Two weeks later, the second immunization was performed with incomplete Freund's adjuvant emulsified at a dose of 30 μg / mouse. After the second immunization, tail blood was collected and serum titer was determined by gradient dilution using ELISA. The results determined whether to boost immunization and the mice with the highest antibody titer were selected for cell fusion.

[0033] (2) Cell fusion: Myeloma cells were sp2 / 0 from BALB / c, which were in the logarithmic growth phase at the time of fusion. The spleen of immunized mice was taken to prepare a single-cell suspension of lymphocytes. Mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:5-1:10, and 1 mL of 50% PEG (pH 8.0) preheated at 37°C was added dropwise. Incomplete culture medium and stop solution were added. After centrifugation and discarding the supernatant, HAT culture medium was added to suspend and mix. The MC volume was adjusted to 50 mL, and the cells were dispensed into 3.5 cm culture dishes, placed in a humidified box, and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0034] (3) Screening and cloning: Select hybridoma cell clones within 7-10 days of fusion, and use purified recombinant BCOR protein for ELISA testing. Mark the cell line number. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 5-6 days after each limiting dilution, and select the monoclonal wells with higher OD280 positive values ​​for limiting dilution until the ELISA results of the entire 96-well plate are positive. Select the monoclonal clone with a high positive value and identify it. The corresponding fusion plate cell line is OTI3D11.

[0035] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI3D11 was cultured in a 10 cm culture dish using DMEM medium containing 15% serum and expanded to approximately 410 μg / mL. 7 When the cell density is about 310 cells, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2L spinner bottle, add serum-free medium, and make the cell density about 310 5 After 1-2 weeks of continuous culture, when the cell death rate reaches 60%-70% (the cell density is about 1-210 6 / ml), collect the cell suspension and centrifuge at 6000 rpm for 20 minutes, take the supernatant, purify the supernatant by affinity chromatography, select the corresponding column material for purification according to the antibody subtype (the subtype is IgG2b, and protein G column material is used for purification). Determine the concentration of the purified anti-human BCOR protein monoclonal antibody, freeze-dry and package (100μg / tube), and finally store at -20℃.

[0036] Example 4 Analysis of variable region genes and amino acid sequences of anti-human BCOR protein monoclonal antibodies Purchase SMARTer from Takara Bio USA ® RACE 5' / 3' kit uses 5'RACE (Rapid Amplification of cDNA Ends) technology to amplify the variable region light chain and heavy chain gene sequences of functional antibodies in hybridoma cells. For the specific experimental process, please refer to Takara Bio USA's SMARTer ® RACE 5' / 3'Kit User Manual.

[0037] Since the antibody is of IgG2b subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of its Ig and Kappa constant regions were designed. The primer sequences are as follows: pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11) pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12) mRNA was extracted from the hybridoma cell line OTI3D11, reverse transcribed into cDNA, and RACE was used to amplify the DNA fragments of the heavy and light chains of the anti-human BCOR protein monoclonal antibody. The amplified light and heavy chains were respectively connected to the cloning vector PUC119 by enzyme digestion, and positive clones were picked by blue-white staining, and the positive plasmids were purified for sequencing using the sequencer ABI 3730, and the sequencing primers were universal primers M13F and M13R.

[0038] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO:13) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO:14) Using the Internet, IMGT / V-QUEST analysis software was used at http: / / www.imgt.org to perform data analysis on the sequencing results of the nucleotide sequences of the light chain and heavy chain of the anti-human BCOR protein monoclonal antibody, respectively, and the amino acid sequence of the light chain variable region of the anti-human BCOR protein monoclonal antibody was obtained as shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID NO.7.

[0039] The total length of the light chain variable region is 109 amino acids, the number of amino acids in the four domains of FR are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR are 12, 3 and 4 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-38aa, 56aa-58aa and 95aa-98aa respectively, and their amino acid sequences are QSLFNSRTRKNY (SEQ ID NO.4), DTS (SEQ ID NO.5) and KQSY (SEQ ID NO.6), respectively.

[0040] The total length of the heavy chain variable region is 113 amino acids, the number of amino acids in the four domains of FR are 24, 17, 36 and 11 respectively, the number of amino acids in the three domains of HCDR are 8, 8 and 9 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-102aa respectively, and their amino acid sequences are GFSIKDTY (SEQ ID NO.8), IDPANGDT (SEQ IDNO.9), DYRGEWFVY (SEQ ID NO.10), respectively.

[0041] Example 5 Immunohistochemical detection kit containing anti-human BCOR protein monoclonal antibody An immunohistochemical detection kit containing an anti-human BCOR protein monoclonal antibody comprises an antigen repair solution [1mM EDTA, 10mM Tris buffer (pH8.0)], the anti-human BCOR protein monoclonal antibody prepared and purified in Example 3, an endogenous peroxidase blocker (hydrogen peroxide), a supersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, a DAB substrate buffer, a DAB concentrated color developing solution, and a hematoxylin staining solution.

[0042] Example 6 Immunohistochemical detection using anti-human BCOR protein monoclonal antibody as primary antibody The immunohistochemical detection kit in Example 5 was used to detect BCOR protein in tissues, and the specific steps are as follows.

[0043] (1) Formalin-fixed endometrial stromal sarcoma tissue blocks were obtained and embedded in paraffin. The sections were sliced ​​using a Leica tissue slicer with a tissue thickness of 4 μm.

[0044] (2) Dewaxing and hydration: analytical grade xylene for 10 min 3 times, anhydrous ethanol for 1 min 3 times, 95% ethanol for 1 min, 85% ethanol for 1 min, 75% ethanol for 1 min, and deionized water for 2 min 3 times.

[0045] (3) Antigen repair: Add antigen repair solution and perform high-pressure heat repair in a pressure cooker for 3 min. When the temperature of the pressure cooker drops to about 90°C, open the pressure cooker, take out the slices, and then cool them naturally to room temperature. Soak them in deionized water for 2 min 3 times.

[0046] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in tissues, let stand at room temperature for 15 min, and soak in deionized water for 2 min 3 times.

[0047] (5) Use an immunohistochemistry pen to draw a frame around the tissue and wash once with 0.1% PBST for 2 min.

[0048] (6) Incubation with primary antibody: Add 200 μl (concentration: 0.39 μg / mL) of diluted anti-human BCOR protein monoclonal antibody secreted by hybridoma cell line OTI3D11 and place in a wet box and incubate at 37°C for 60 min. Wash with 0.1% PBST for 2 min 3 times.

[0049] (7) Incubation with secondary antibody: Add 100 μl of secondary antibody PV-8000 and incubate at 37°C for 30 min. Wash with 0.1% PBST for 2 min 3 times.

[0050] (8) DAB color development: Add 120 μl of DAB color development solution, let stand at room temperature for 5 min, rinse under running water to stop color development, and rinse with tap water three times.

[0051] (9) Hematoxylin re-staining, differentiation and blueing: Place in hematoxylin solution for 10-12 seconds, rinse with tap water three times to stop coloring, differentiate in 1% hydrochloric acid ethanol solution, rinse with tap water three times to stop, place in freshly boiled Tris-EDTA disodium solution at pH 8.0 to blue, then place in room temperature Tris-EDTA disodium solution at pH 9.0 for a few seconds, rinse with tap water three times. Observe the staining under a microscope. If it is normal, stop and recover the hematoxylin staining solution; if it is too differentiated, repeat the above steps until the staining is qualified.

[0052] (10) Dehydration and transparency: 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min 3 times, xylene for 1 min 3 times, and sealing with neutral gum.

[0053] (11) Microscopic examination, such as Figure 3 shown.

[0054] The anti-human BCOR protein monoclonal antibody secreted by hybridoma cell OTI3D11 was used to detect endometrial stromal sarcoma tissue by IHC. The tumor cells indicated by the star symbol (only for example) were arranged in oval and short spindle sheets. After IHC detection, they were brown and located in the cell nucleus, which was consistent with the theoretical subcellular localization of BCOR, indicating that the tumor cells in this tissue sample expressed BCOR protein. The vascular endothelium indicated by the arrow (only for example), as well as the infiltrating lymphocytes and other stromal cells, did not have positive staining, indicating that there was no expression of BCOR protein. The results were consistent with the clinical diagnosis information of this case. The concentration of the monoclonal antibody used in this test result was 0.39μg / mL. Under this condition, the positive signal intensity on the tumor cells met the clinical judgment requirements, and there was no background staining that affected the diagnosis, indicating that the antibody secreted by hybridoma cell OTI3D11 had good specificity and high sensitivity.

[0055] The above results show that the anti-human BCOR protein monoclonal antibody secreted by the hybridoma cell line OTI3D11 described in the present application has high specificity and sensitivity. Therefore, the anti-BCOR monoclonal antibody secreted by the hybridoma cell line OTI3D11 can be used for immunohistochemical detection to detect the expression of BCOR in related tumors, providing a basis for clinicians to diagnose patients.

[0056] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A monoclonal antibody or antigen-binding fragment against human BCOR protein, characterized in that: The monoclonal antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the amino acid sequence of LCDR1 comprises QSLFNSRTRKNY, the amino acid sequence of LCDR2 comprises DTS, and the amino acid sequence of LCDR3 comprises KQSY.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that The heavy chain variable region comprises HCDR1-3, the amino acid sequence of the HCDR1 comprises GFSIKDTY, the amino acid sequence of the HCDR2 comprises IDPANGDT, and the amino acid sequence of the HCDR3 comprises DYRGEWFVY.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.

3.

4. The monoclonal antibody or antigen-binding fragment according to claim 2, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.

7.

5. A hybridoma cell line capable of secreting anti-human BCOR protein monoclonal antibodies, characterized in that: The hybridoma cell line is the mouse anti-human BCOR monoclonal hybridoma cell line OTI3D11, whose preservation number is CGMCC No.46244, and it was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 28, 2024.

6. A monoclonal antibody or antigen-binding fragment against human BCOR protein, characterized in that: The monoclonal antibody or antigen-binding fragment against human BCOR protein is obtained by secretion from the hybridoma cell line according to claim 5.

7. Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4 or claim 6, characterized in that: The application is any of the following, 1) Labeling of human BCOR protein in cells or tissues; 2) Prepare an immunohistochemical detection kit for human BCOR protein.

8. An immunohistochemical detection kit for human BCOR protein, characterized in that: The immunohistochemical detection kit comprises a primary antibody detection reagent, and the primary antibody detection reagent contains the monoclonal antibody or antigen fragment according to any one of claims 1 to 4 or claim 6.

9. The kit according to claim 8, characterized in that The kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color developing solution, and hematoxylin staining solution.

10. The kit according to claim 9, characterized in that The ultrasensitive secondary antibody reagent is an ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

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