Monoclonal Antibody Against Human YAP1 Protein, Hybridoma Cell Line Thereof and Application

Abstract

This application relates to the field of immunoglobulin technology, specifically to anti-human YAP1 protein monoclonal antibodies, hybridoma cell lines, and applications thereof. This application provides an anti-human YAP1 protein monoclonal antibody secreted and produced by the hybridoma cell line OTI4G8. The hybridoma cell line OTI4G8 was deposited with the China General Microbiology Center (CGMCC) on November 28, 2024, with the deposit number CGMCC No. 46246. The hybridoma cell line OTI4G8 provided herein can stably secrete anti-human YAP1 protein monoclonal antibodies that specifically bind to the YAP1 protein, significantly improving the specificity, accuracy, and reliability of YAP1 protein immunoassays and making it suitable for labeling 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), encoded by the YAP1 gene, is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes gene and exhibits protein tyrosine kinase activity, acting as a transcriptional coactivator. It is primarily composed of a TID domain, a WW domain, and a TAD domain. Some isoforms contain SH3-BM and leucine zinc finger structures. Because it lacks a DNA binding domain, its transcriptional regulatory function relies on interactions with several other proteins, such as transcription factors (such as TEAD, RUNX2, and SMAD) and chromatin remodeling proteins. It plays a vital role in human development, growth, DNA repair, and endogenous homeostasis. Studies have shown that activated YAP1 promotes tissue proliferation, differentiation, and regeneration, contributing to wound repair. Normally activated, YAP1 forms a complex with TAZ and 14-3-3 proteins, localizing to the cytoplasm and initiating apoptosis. Hyperactivated YAP1 significantly enhances tissue cell proliferation, thereby promoting tumor formation. In tumor cells, YAP1 exists in a phosphorylated form, thus enhancing the anti-apoptotic ability of tumor cells. 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 serves as a potential tumor marker. Targeting YAP1 may be a promising approach for future cancer treatment. Studies have shown that YAP1 is highly expressed in all meningiomas, regardless of histological subtype and grade. Immunohistochemical staining consistently shows high expression in the nucleus, with greater variability in cytoplasmic staining. In triple-negative breast cancer, the receptor tyrosine kinase Met can induce YAP expression. In laryngeal and gastric cancer cells, YAP has also been found to enhance tumor cell proliferation and invasion by upregulating β-catenin pathway activity. YAP1 is associated with drug sensitivity in various cancers, including non-small cell lung cancer and small cell lung cancer. In NSCLC cell lines, YAP1 overexpression promotes resistance to the chemotherapeutic drugs paclitaxel and cisplatin, as well as the targeted drugs erlotinib / gefitinib. High YAP1 expression is a marker of poor prognosis in various tumors, including non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, breast cancer, and papillary thyroid carcinoma.

[0004] Currently, 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 for a variety of 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 uses 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 terminal modification 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 number of amino acids in the four FR domains are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR1-3 are 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 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, preferably, an amino acid sequence with 95%, 96%, 97%, 98% or 99% homology.

[0010] Furthermore, the full length of the heavy chain variable region is 108 amino acids, the amino acid numbers of the four domains of 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 the mouse anti-human YAP1 monoclonal hybridoma cell line OTI4G8, whose deposit number is CGMCC No. 46246, and deposited on November 28, 2024 in the General Microbiology Center of the China Culture Collection Administration (CGMCC), and the depository address 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 containing 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 development solution, and hematoxylin staining solution.

[0017] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution; preferably, the secondary antibody reagent is ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0018] Preservation Information

[0019] Biomaterial: OTI4G8

[0020] Classification and nomenclature: Mouse anti-human YAP1 monoclonal hybridoma cell line

[0021] Deposit number: CGMCC No.46246

[0022] Deposit date: November 28, 2024

[0023] Depository: China General Microbiology Center (CGMCC)

[0024] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Compared to the prior art, the anti-human YAP1 protein monoclonal antibodies or antigen-binding fragments provided in this application are capable of specifically binding to human YAP1 protein, significantly improving the specificity, accuracy, and reliability of YAP1 protein immunoassays. Furthermore, the hybridoma cell line OTI4G8 provided in this application can stably secrete anti-human YAP1 protein monoclonal antibodies. Secondly, using the anti-human YAP1 protein monoclonal antibodies and kits in this application, IHC assays for lung cancer, liver cancer, and other tissues clearly detected YAP1 protein expression in cells, demonstrating that the anti-human YAP1 protein monoclonal antibodies in this application are well suited for immunohistochemical detection of YAP1 protein in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation of the present application, the following is a brief introduction to the drawings required for describing the specific implementation.

[0027] Figure 1 This is a graph showing the results of Western blot detection of recombinant YAP1 protein in Example 2. The expression of recombinant YAP1 protein in E. coli cells was detected using an anti-HIS antibody. Lane L shows the detection result using E. coli cell lysate transfected with an empty vector as the antigen, and lane R shows the detection result using E. coli cell lysate transfected with the pET23a-rYAP1 plasmid as the antigen.

[0028] Figure 2 This is a diagram showing the SDS-PAGE results of YAP1 protein in Example 2. 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.

[0029] 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.

[0030] Figure 4This 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.

[0031] Figure 5 This 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

[0032] The present application will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0033] Example 1 Construction of YAP1 protein recombinant expression plasmid

[0034] The YAP1 gene NM_001130145 was selected from Genebank. Based on the amino acid sequence characteristics of YAP1, a fragment of amino acids 37-321 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, and restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene. The fragment was inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rYAP1.

[0035] Example 2 Expression and purification of recombinant YAP1 protein

[0036] (1) Transformation of E. coli cells: Thaw 100 μl of competent cells on ice, then add the recombinant plasmid DNA and mix gently. After ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, and then continue to ice-bath for 1-2 minutes. Add 500 μl of fresh antibiotic-free LB medium in a clean bench, incubate on a shaker at 37°C for 45 minutes, and then evenly spread an appropriate amount of bacterial solution on a plate containing antibiotics. Place the culture dish upside down in a 37°C constant temperature incubator and culture overnight.

[0037] (2) Cell lysis: 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 them for 20 minutes, and centrifuge them at 12000 rpm at 4°C for 20 minutes. Collect the supernatant. Take a small amount of supernatant protein and use anti-His antibody for Western blotting. Figure 1 .

[0038] Depend on Figure 1The results showed that the E. coli cell lysate transfected with the pET23a-rYAP1 plasmid was located in lane R, with an obvious specific band at 25kD, while the control lysate transformed with the empty vector had no band of the corresponding size in lane 1, indicating that the cells specifically expressed the recombinant YAP1 protein.

[0039] (3) Nickel affinity chromatography column purification: Equilibrate the nickel column with buffer, filter the supernatant with a 0.45 μm filter membrane, load the sample, collect the outflow, elute with buffer to remove unbound proteins, and finally elute with eluent containing different concentrations of imidazole. After collection, the eluted proteins that meet the requirements are combined and 10% glycerol is added. The purified recombinant YAP1 protein is identified by SDS-PAGE electrophoresis, see Figure 2 .

[0040] 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 immunogen amino acid fragment, indicating that a recombinant YAP1 protein with good purity had been obtained.

[0041] Example 3 Preparation and screening of OTI4G8-secreted monoclonal antibodies against human YAP1 protein

[0042] 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:

[0043] (1) Animal immunization: Purified YAP1 antigen was emulsified in 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, a second immunization was performed using incomplete Freund's adjuvant emulsified at a dose of 30 μg / mouse. After the second immunization, tail blood was collected and serum titers were determined by serial dilution ELISA. Based on the results, the decision to boost immunization was made, and the mouse with the highest antibody titer was selected for cell fusion.

[0044] (2) Cell fusion: Myeloma cells were sp2 / 0 derived from BALB / c cells, 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, and the supernatant was discarded after centrifugation. HAT culture medium was added to suspend and mix thoroughly. The MC volume was adjusted to 50 mL, and the cells were divided into 3.5 cm culture dishes, placed in a humidified box, and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0045] (3) Screening and cloning: Within 7-10 days of fusion, hybridoma cell clones were selected and tested by ELISA using purified recombinant YAP1 protein. The cell line number was marked. The cells in the positive wells were subjected to limiting dilution. The ELISA value was measured 5-6 days after each limiting dilution. The monoclonal wells with higher OD280 positive values were selected for limiting dilution until the ELISA results of the entire 96-well plate were positive. The monoclonal wells with higher positive values were selected for identification. The corresponding fusion plate cell line was OTI4G8.

[0046] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI4G8 was cultured in a 10 cm culture dish using DMEM containing 15% serum and expanded to approximately 410 7 When the cells reached 310, centrifuge at 800 rpm for 5 min, discard the supernatant, transfer the cells to a 2 L spinner flask, add serum-free medium, and adjust the cell density to approximately 310. 5 After 1-2 weeks of culture, when the cell death rate reaches 60%-70% (the cell density is about 1-210 6 The cell suspension was centrifuged at 6000 rpm for 20 minutes. The supernatant was purified by affinity chromatography using a column selected based on the antibody subtype (IgG1, protein G column). The purified anti-human YAP1 monoclonal antibody was concentrated, lyophilized, and aliquoted (100 μg / tube) for storage at -20°C.

[0047] Example 4 Analysis of variable region genes and amino acid sequences of anti-human YAP1 protein monoclonal antibodies

[0048] Purchase SMARTer from Takara Bio USA ® The 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 detailed experimental procedures, please refer to the SMARTer ® RACE 5' / 3'Kit User Manual.

[0049] Since the antibody is of IgG1 subtype, specific gene primers pRace-H-GSP and pRace-K-GSP were designed for the 3' end of its Ig and Kappa constant regions. The primer sequences are as follows:

[0050] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11)

[0051] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12)

[0052] mRNA was extracted from the hybridoma cell line OTI4G8 and reverse-transcribed into cDNA. DNA fragments of the heavy and light chains of the anti-human YAP1 monoclonal antibody were amplified by RACE. The amplified light and heavy chains were ligated into the cloning vector PUC119 via enzyme digestion. Positive clones were picked by blue-white staining, and the plasmids were purified and sequenced using an ABI 3730 sequencer with universal primers M13F and M13R.

[0053] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO:13) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO:14)

[0054] The nucleotide sequence of the light chain and heavy chain of the anti-human YAP1 protein monoclonal antibody was analyzed using IMGT / V-QUEST analysis software on the Internet at http: / / www.imgt.org. The amino acid sequence of the light chain variable region of the anti-human YAP1 protein monoclonal antibody was shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID NO. 7.

[0055] The total length of the light chain variable region is 109 amino acids, with the four FR domains containing 26, 17, 36 and 11 amino acids respectively, the three LCDR domains containing 11, 3 and 5 amino acids respectively, the LCDR1, LCDR2 and LCDR3 regions containing 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).

[0056] The full length of the heavy chain variable region is 108 amino acids, with the amino acid numbers of the four FR domains being 24, 17, 36 and 11 respectively, the amino acid numbers of the three HCDR domains being 8, 8 and 4 respectively, HCDR1, HCDR2 and HCDR3 being 25aa-32aa, 50aa-57aa and 94aa-97aa respectively, and their amino acid sequences being: GYAFSSYW (SEQ ID NO.8), IHPGDGEP (SEQ ID NO.9), ANSG (SEQ ID NO.10).

[0057] Example 5 Immunohistochemistry detection kit containing anti-human YAP1 protein monoclonal antibody

[0058] An immunohistochemical detection kit containing an anti-human YAP1 protein monoclonal antibody comprises an antigen retrieval solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], the anti-human YAP1 protein monoclonal antibody prepared and purified in Example 3, an endogenous peroxidase blocker (hydrogen peroxide), a hypersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, a DAB substrate buffer, a DAB concentrated color development solution, and a hematoxylin staining solution.

[0059] Example 6 Immunohistochemical Detection Using Anti-Human YAP1 Protein Monoclonal Antibody as Primary Antibody

[0060] The immunohistochemical detection kit in Example 5 was used to detect YAP1 protein in tissues, and the specific steps are as follows.

[0061] (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.

[0062] (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.

[0063] (3) Antigen repair: Add antigen repair solution and perform high-pressure heat repair in a pressure cooker for 3 minutes. 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 in deionized water for 2 minutes three times.

[0064] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in the tissue, let it stand at room temperature for 15 minutes, and soak it in deionized water for 2 minutes three times.

[0065] (5) Use an immunohistochemistry pen to draw a frame around the tissue and wash once with 0.1% PBST for 2 minutes.

[0066] (6) Incubation with primary antibody: Add 200 μl of diluted anti-human YAP1 monoclonal antibody secreted by hybridoma cell line OTI4G8 and incubate in a humidified chamber at 37°C for 60 min. Wash with 0.1% PBST for 2 min three times.

[0067] (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 three times.

[0068] (8) DAB color development: Add 120 μl of DAB color development solution, let it stand at room temperature for 5 min, rinse under tap water to stop color development, and rinse with tap water three times.

[0069] (9) Hematoxylin counterstaining, differentiation, and blueing: Place the sample in the hematoxylin solution for 10-12 seconds, rinse three times with tap water to stop the color development, differentiate in a 1% hydrochloric acid ethanol solution, rinse three times with tap water to stop the color development, place in a freshly boiled Tris-EDTA disodium solution at pH 8.0 to blue the sample, then place in a room temperature Tris-EDTA disodium solution at pH 9.0 for a few seconds, and rinse three times with tap water. Observe the staining under a microscope. If the staining is normal, stop and recover the hematoxylin staining solution. If the staining is excessive, repeat the above steps until the staining is acceptable.

[0070] (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.

[0071] (11) Microscopic examination, e.g. Figure 3-Figure 5 shown.

[0072] 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 Figures a and b show four lung cancer tissues from different sources. Images a and b show YAP1-expressing lung cancers, with varying degrees of YAP1 expression in the tumor cells, with staining localized in the cytoplasm and nucleus. Images c and d show YAP1-deficient lung cancer tissues. The tumor cells indicated by arrows (for example only) lack brown staining and are negative, indicating no YAP1 protein expression. Vascular endothelial cells and other stromal cells, serving as internal controls, show positive expression. Figure 4 These are liver cancer cells. The staining results show that the cancer cells are stained in the cytoplasm and nucleus, and the bile duct epithelium is positively expressed. Figure 5 The staining results of kidney tissue show positive expression in both glomeruli and renal tubules.

[0073] These results demonstrate that the anti-human YAP1 monoclonal antibody secreted by the hybridoma cell line OTI4G8 described herein has high specificity. Therefore, the anti-YAP1 monoclonal antibody secreted by the hybridoma cell line OTI4G8 can be used in immunohistochemistry to detect YAP1 expression in related tumors, providing a reference for clinicians in patient diagnosis, medication, and prognosis.

[0074] 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 as long as they are within the scope of the claims of the present application, they are protected by the patent law.

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 are QSLLWSNGKTY, STS, and VQATH, respectively; and the heavy chain variable region comprises HCDR1-3, and the amino acid sequences of HCDR1-3 are GYAFSSYW, IHPGDGEP, and ANSG, respectively.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

3.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein The heavy chain variable region comprises 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 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 from 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) Preparation of labeling reagents for 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-binding 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 development solution, and hematoxylin staining solution.

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