Anti-human YAP1 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 YAP1 protein monoclonal antibody and a hybridoma cell strain and application thereof. The invention provides an anti-human YAP1 protein monoclonal antibody which is secreted by a hybridoma cell strain OTI4G8, the hybridoma cell strain OTI4G8 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 28, 2024, and the preservation number of the hybridoma cell strain OTI4G8 is CGMCC No.46246. The invention further provides a preparation method of the anti-human YAP1 protein monoclonal antibody. The hybridoma cell strain OTI4G8 provided by the invention can stably secrete the anti-human YAP1 protein monoclonal antibody, and can be specifically combined with the YAP1 protein, so that the specificity, accuracy and reliability of YAP1 protein immunodetection are remarkably improved, and the hybridoma cell strain OTI4G8 can be suitable for marking the YAP1 protein in tissues.

Description

Technical Field

[0001] The present application relates to the technical field of immunoglobulins, and in particular to anti-human YAP1 protein monoclonal antibodies and hybridoma cell lines and applications thereof. Background Art

[0002] YAP1 (Yes-Associated Protein 1) is the encoded product of the YAP1 gene. It is a proline-rich phosphoprotein that can bind to the SH3 domain of the Yes gene and has protein tyrosine kinase activity. It is a transcriptional coactivator. It is mainly composed of the TID domain, WW domain, and TAD domain. Some isoforms contain SH3-BM and leucine zinc finger structures. Since it does not contain a DNA binding domain, its transcriptional regulatory function depends on the interaction with several other proteins, such as transcription factors (such as TEAD, RUNX2, SMAD) and chromatin remodeling proteins. It plays an active role in human development, growth, DNA repair, and endogenous homeostasis. Studies have found that YAP1 in an activated state can promote tissue proliferation, differentiation, and regeneration, which has a positive effect on the repair of wounds in organisms. YAP1 in a normally activated state can form a complex with TAZ and 14-3-3 proteins, localize in the cytoplasm, and initiate cell apoptosis. YAP1 in a hyperactivated state will greatly promote the proliferation ability of tissue cells, thereby promoting the formation of tumors. In tumor cells, YAP1 exists in a phosphorylated form, so the anti-apoptosis ability of tumor cells is enhanced. YAP1 can also interact with catenin and Kras to regulate the metastasis and invasion ability of tumor cells.

[0003] YAP1 is highly expressed in many tumors and is a potential tumor marker. Targeting YAP1 may be a direction for future tumor treatment. Studies have shown that YAP1 is highly expressed in all meningiomas. Regardless of the histological subtype and grade, the distribution of immunohistochemical staining in the cell nucleus is always high, and the staining in the cytoplasm varies greatly. In triple-negative breast cancer, the receptor tyrosine kinase Met can induce the expression of YAP. In laryngeal cancer cells and gastric cancer cells, YAP was also found to enhance the proliferation and invasion of tumor cells by upregulating the activity of the β-catenin pathway. YAP1 is associated with the drug sensitivity of many cancers, including non-small cell lung cancer and small cell lung cancer. In NSCLC cell lines, overexpression of YAP1 promotes tumor cell resistance to the chemotherapy drugs paclitaxel, cisplatin, and targeted drugs erlotinib / gefitinib. High expression of YAP1 is a sign of poor prognosis in many tumors, including non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, breast cancer, and papillary thyroid carcinoma.

[0004] Currently available YAP1 inhibitors have strong anti-tumor activity, so it is crucial to develop highly specific and sensitive monoclonal antibodies that recognize YAP1 proteins to guide medication and prognosis of various tumors. Summary of the invention

[0005] In view of this, the present application provides an anti-human YAP1 protein monoclonal antibody, a hybridoma cell line capable of secreting the anti-human YAP1 protein monoclonal antibody, and applications of the anti-human YAP1 protein monoclonal antibody and hybridoma cell line.

[0006] On the one hand, the present application provides a monoclonal antibody or antigen-binding fragment against human YAP1 protein, wherein 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, and the amino acid sequences of the LCDR1-3 sequentially include QSLLWSNGKTY (SEQ ID NO.4), STS (SEQ ID NO.5), and VQATH (SEQ ID NO.6), and the heavy chain variable region comprises HCDR1-3, and the amino acid sequences of the HCDR1-3 sequentially include GYAFSSYW (SEQ ID NO.8), IHPGDGEP (SEQ ID NO.9), and ANSG (SEQ ID NO.10).

[0007] Furthermore, 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 95% 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 95%, 96%, 97%, 98% or 99% homology.

[0008] 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 11, 3 and 5 respectively, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-37aa, 55aa-57aa and 94aa-98aa respectively.

[0009] Furthermore, 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 95% 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 95%, 96%, 97%, 98% or 99% homology.

[0010] Furthermore, the total length of the heavy chain variable region is 108 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 4 respectively, and the HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-97aa respectively.

[0011] On the other hand, the present application also provides a hybridoma cell line capable of secreting anti-human YAP1 protein monoclonal antibodies or antigen-binding fragments, the hybridoma cell line being a mouse anti-human YAP1 monoclonal hybridoma cell line OTI4G8, whose deposit number is CGMCC No.46246, and which was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on November 28, 2024. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] On the other hand, the present application also provides a monoclonal antibody or antigen-binding fragment against human YAP1 protein, wherein the monoclonal antibody or antigen-binding fragment against human YAP1 protein is obtained by secretion of the hybridoma cell line OTI4G8.

[0013] 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 YAP1 protein in cells or tissues; 2) preparing an immunohistochemical detection kit for human YAP1 protein.

[0014] Furthermore, the cells are cancer cells or normal tissue cells, preferably lung cancer, liver cancer or kidney tissue.

[0015] On the other hand, the present application also provides an immunohistochemical detection kit for human YAP1 protein, wherein the immunohistochemical detection kit comprises an antibody detection reagent, and the antibody detection reagent contains the monoclonal antibody or antigen fragment.

[0016] 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.

[0017] 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.

[0018] Preservation Information Biomaterial: OTI4G8 Classification and naming: Mouse anti-human YAP1 monoclonal hybridoma cell line Deposit number: CGMCC No.46246 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.

[0019] Compared with the prior art, the anti-human YAP1 protein monoclonal antibody or antigen-binding fragment provided in the present application can specifically bind to the human YAP1 protein, significantly improving the specificity, accuracy and reliability of YAP1 protein immunoassay; at the same time, the hybridoma cell line OTI4G8 provided can also stably secrete anti-human YAP1 protein monoclonal antibodies. Secondly, using the anti-human YAP1 protein monoclonal antibody and kit of the present application, the IHC method is used to detect lung cancer, liver cancer and other tissues, and the YAP1 protein expressed in the cells can be clearly detected, indicating that the anti-human YAP1 protein monoclonal antibody of the present application can be well used for immunohistochemical detection of YAP1 protein in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 This is a graph showing the results of Western blot detection of the recombinant YAP1 protein in Example 2, in which the expression of the recombinant YAP1 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-rYAP1 plasmid as the antigen.

[0022] Figure 2 This is a diagram of the SDS-PAGE result of the YAP1 protein in Example 2. The recombinant YAP1 protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining.

[0023] Figure 3 This is a schematic diagram of the results of IHC detection of lung cancer using the monoclonal antibody secreted by the hybridoma cell line OTI4G8 in Example 6.

[0024] Figure 4 This is a schematic diagram of the results of IHC detection of liver cancer using the monoclonal antibody secreted by the hybridoma cell line OTI4G8 in Example 6.

[0025] Figure 5This is a schematic diagram of the results of IHC detection of kidneys using the monoclonal antibody secreted by the hybridoma cell line OTI4G8 in Example 6. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1 Construction of YAP1 protein recombinant expression plasmid YAP1 gene NM_001130145 was selected from Genebank. According to the amino acid sequence characteristics of YAP1, the 37-321 amino acid fragment of YAP1 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 inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rYAP1.

[0028] Example 2 Expression and purification of recombinant YAP1 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.

[0029] (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 .

[0030] Depend on Figure 1 The results showed that the E. coli cell lysate transfected with pET23a-rYAP1 plasmid was located in lane R, with an obvious specific band at 25kD, while there was no band of the corresponding size in lane 1 of the control lysate transformed with an empty vector, indicating that the cells specifically expressed the recombinant YAP1 protein.

[0031] (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 effluent was collected. The unbound protein was removed by elution with buffer, and finally eluted with eluent containing different concentrations of imidazole. After collection, the eluted proteins that met the requirements were combined and 10% glycerol was added. The purified recombinant YAP1 protein was identified by SDS-PAGE electrophoresis, see Figure 2 .

[0032] Depend on Figure 2 The results showed that the purified protein had an obvious specific band at 25kD 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 YAP1 protein with good purity had been obtained.

[0033] Example 3 Preparation and screening of OTI4G8 secreted anti-human YAP1 protein monoclonal antibodies BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized with purified recombinant YAP1 protein (hereinafter referred to as YAP1 antigen) according to standard methods. The specific method is as follows: (1) Animal immunization: The purified YAP1 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 decision on whether to boost immunization was made based on the results, and the mouse with the highest antibody titer was selected for cell fusion.

[0034] (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.

[0035] (3) Screening and cloning: Select hybridoma cell clones within 7-10 days of fusion, and use purified recombinant YAP1 protein for ELISA test. 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 result of the whole 96-well plate is positive. Select the monoclonal clone with high positive value for finalization, and its corresponding fusion plate cell line is OTI4G8.

[0036] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI4G8 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, and select the corresponding column material for purification according to the antibody subtype (the subtype is IgG1, and protein G column material is used for purification). The concentration of the purified anti-human YAP1 protein monoclonal antibody is measured, lyophilized and packaged (100μg / tube), and finally stored at -20℃.

[0037] Example 4 Analysis of variable region genes and amino acid sequences of anti-human YAP1 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.

[0038] Since the antibody is of IgG1 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 hybridoma cell line OTI4G8, reverse transcribed into cDNA, and RACE was used to amplify the DNA fragments of the heavy and light chains of the anti-human YAP1 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.

[0039] 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 YAP1 protein monoclonal antibody, respectively, to obtain the amino acid sequence of the light chain variable region of the anti-human YAP1 protein monoclonal antibody as shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.7.

[0040] The total length of the light chain variable region is 109 amino acids, the amino acid numbers of the four domains of FR are 26, 17, 36 and 11 respectively, the amino acid numbers of the three domains of LCDR are 11, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-37aa, 55aa-57aa and 94aa-98aa respectively, and their amino acid sequences are: QSLLWSNGKTY (SEQ ID NO.4), STS (SEQ ID NO.5), VQATH (SEQ ID NO.6).

[0041] The total length of the heavy chain variable region is 108 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 4 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-97aa respectively, and their amino acid sequences are: GYAFSSYW (SEQ ID NO.8), IHPGDGEP (SEQ IDNO.9), ANSG (SEQ ID NO.10).

[0042] Example 5 Immunohistochemistry detection kit containing anti-human YAP1 protein monoclonal antibody An immunohistochemical detection kit containing an anti-human YAP1 protein monoclonal antibody comprises an antigen repair solution [1mM EDTA, 10mM Tris buffer (pH8.0)], the anti-human YAP1 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.

[0043] Example 6 Immunohistochemical detection using anti-human YAP1 protein monoclonal antibody as primary antibody The immunohistochemical detection kit in Example 5 was used to detect YAP1 protein in tissues, and the specific steps are as follows.

[0044] (1) Formalin-fixed lung cancer, liver cancer, and kidney tissue blocks were paraffin-embedded and sliced ​​using a Leica tissue slicer with a tissue thickness of 4 μm.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] (5) Use an immunohistochemistry pen to draw a frame around the tissue and wash once with 0.1% PBST for 2 min.

[0049] (6) Incubation with primary antibody: Add 200 μl of diluted anti-human YAP1 monoclonal antibody secreted by hybridoma cell line OTI4G8 and place in a wet box, incubate at 37°C for 60 min. Wash with 0.1% PBST for 2 min 3 times.

[0050] (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.

[0051] (8) DAB color development: Add 120 μl of DAB color development solution, let stand at room temperature for 5 min, rinse under tap water to stop color development, and rinse three times with tap water.

[0052] (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.

[0053] (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.

[0054] (11) Microscopic examination, such as Figure 3-Figure 5 shown.

[0055] The anti-human YAP1 protein monoclonal antibody secreted by hybridoma cell OTI4G8 was used to detect lung cancer, liver cancer and kidney tissues by IHC method. Figure 3 These are 4 lung cancer tissues from different sources, of which a and b are lung cancers expressing YAP1. There are different degrees of YAP1 expression in tumor cells, and the staining is localized in the cytoplasm and nucleus. c and d are lung cancer tissues lacking YAP1 expression. The tumor cells indicated by the arrows (for example only) are not stained brown and are negative, indicating that there is no expression of YAP1 protein. The vascular endothelial cells and other stromal cells that can be used as internal controls show positive expression. Figure 4 These are liver cancer cells. The staining results showed that the cancer cells showed cytoplasmic and nuclear staining, and the bile duct epithelium showed positive expression. Figure 5 This is the staining result of kidney tissue, which shows that both glomeruli and renal tubules are positively expressed.

[0056] The above results show that the anti-human YAP1 protein monoclonal antibody secreted by the hybridoma cell line OTI4G8 described in the present application has high specificity. Therefore, the anti-YAP1 monoclonal antibody secreted by the hybridoma cell line OTI4G8 can be used for immunohistochemical detection to detect the expression of YAP1 in related tumors, providing a reference for clinicians' diagnosis, medication and prognosis of patients.

[0057] 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 non-creative modifications to the present embodiment as needed, but such modifications are protected by 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 YAP1 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, and the amino acid sequences of LCDR1-3 sequentially include QSLLWSNGKTY, STS, and VQATH, and the heavy chain variable region comprises HCDR1-3, and the amino acid sequences of HCDR1-3 sequentially include GYAFSSYW, IHPGDGEP, and ANSG.

2. 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 95% 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.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, 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 95% 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.

4. A hybridoma cell line capable of secreting anti-human YAP1 protein monoclonal antibodies, characterized in that: The hybridoma cell line is the mouse anti-human YAP1 monoclonal hybridoma cell line OTI4G8, whose preservation number is CGMCC No.46246, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on November 28, 2024.

5. A monoclonal antibody or antigen-binding fragment against human YAP1 protein, characterized in that: The anti-human YAP1 protein monoclonal antibody or antigen-binding fragment is obtained by secretion of the hybridoma cell line according to claim 4.

6. Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3 or claim 5, characterized in that: The application is any of the following, 1) Labeling of human YAP1 protein in cells or tissues; 2) Prepare an immunohistochemical detection kit for human YAP1 protein.

7. An immunohistochemical detection kit for human YAP1 protein, characterized in that: The immunohistochemical detection kit comprises an antibody detection reagent, and the antibody detection reagent contains the monoclonal antibody or antigen fragment according to any one of claims 1 to 3 or claim 5.

8. The kit according to claim 7, 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.

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