Canine PD-L1 monoclonal antibody, hybridoma cell strain secreting canine PD-L1 monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a canine PD-L1 monoclonal antibody, a hybridoma cell strain secreting the canine PD-L1 monoclonal antibody and application of the canine PD-L1 monoclonal antibody. The hybridoma cell strain is 3B4C7B2F10C10 preserved in the China Center for Type Culture Collection on June 24, 2025, the preservation number is CCTCC NO: C2025167, the hybridoma cell strain can stably secrete the antibody, and the dog PD-L1 monoclonal antibody can recognize the dog PD-L1 protein, has good color development in dog tumor immunohistochemistry, can accurately detect the expression degree of the PD-L1 in the dog tumor, and can be used for preparing the dog PD-L1 monoclonal antibody. The expression analysis of the PD-L1 has important significance on the clinical application of a PD-1 / PD-L1 inhibitor in dogs, the high expression of the PD-L1 can be used as an important marker for the immunotherapy effect of the dogs, and the expression level of the PD-L1 can be used as an important reference factor for clinical test design and treatment decision in the future. Meanwhile, reference is provided for immunohistochemical auxiliary diagnosis of tumor malignancy degree of dog tumors and dog PD-1 and PD-L1 monoclonal antibody mediated dog tumor immunotherapy prognosis, and the application prospect is huge.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology and genetic engineering antibody technology, in particular to a canine PD-L1 monoclonal antibody, a hybridoma cell strain secreting the canine PD-L1 monoclonal antibody and application thereof. BACKGROUND

[0002] The number of dogs as companion animals has increased dramatically. According to the 2023 China Pet Medical Industry Report Blue Book, it is predicted that the number of dogs and cats in China will increase from 190 million to 210 million in the next five years (from 2023). Cats account for about 55%, and dogs account for about 45%. As the age of dogs increases, tumor diseases have entered a high-incidence period. The incidence adjustment rate of dogs and cats is lower than that of humans (287.3 / 100,000 / year), which is 72.1 / 100,000 / year and 165.7 / 100,000 / year, respectively. About 230,000 dogs and cats are affected each year. For tumor diseases, whether for animals or humans, the main treatment methods are surgical resection, chemotherapy (vincristine, cyclophosphamide, doxorubicin), radiotherapy, and immunotherapy. Current medical immunotherapy includes humanized PD-1 and PD-L1 monoclonal antibody injection therapy and CAR-T, NK, TCR-T cell therapy. Immunotherapy is a new treatment method, which is of great concern due to its small side effects, strong targeting and good effect. The monoclonal antibody treatment method is simple, effective, and has no tumorigenic risk of CAR-T cells.

[0003] Common tumor diseases in dogs include breast cancer, lymphoma, and subcutaneous squamous cell carcinoma. The diagnosis of tumor diseases is mainly diagnosed by immunohistochemical technology. Programmed death ligand-1 (PD-L1) is an important tumor surface marker molecule that is highly expressed on the surface of some tumor cells and can be used as an important target for immunohistochemistry. Currently, there is no commercial kit for detecting PD-L1 protein in canine tumor tissues by immunohistochemistry in the domestic veterinary field, and no drug based on canine PD-L1 monoclonal antibodies for treating canine tumors. SUMMARY

[0004] Different monoclonal antibodies recognize different epitopes, and each monoclonal antibody has a unique epitope recognition characteristic. This invention provides a canine PD-L1 monoclonal antibody, a hybridoma cell line secreting a canine PD-L1 monoclonal antibody, and their applications. The hybridoma cell line obtained through screening for the anti-canine PD-L1 monoclonal antibody can stably secrete the antibody. This PD-L1 monoclonal antibody can recognize canine PD-L1 protein, exhibits good staining in canine tumor immunohistochemistry, and can accurately detect the level of PD-L1 expression in canine tumors. PD-L1 expression analysis is of great significance for the clinical application of PD-1 / PD-L1 inhibitors in dogs. High PD-L1 expression can serve as an important biomarker for the efficacy of canine immunotherapy, supporting the use of PD-L1 expression levels as an important reference factor in future clinical trial design and treatment decisions. It also provides a reference for the immunohistochemical assessment of tumor malignancy and prognosis of immunotherapy in canine tumors, showing great application potential.

[0005] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0006] A hybridoma cell line secreting an anti-canine PD-L1 monoclonal antibody, wherein the hybridoma cell line is 3B4C7B2F10C10, was deposited on June 24, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2025167, located at Wuhan University, Wuhan, China.

[0007] As an improvement, the method for preparing the hybridoma cell line 3B4C7B2F10C10 includes the following steps:

[0008] S1, the clone of the canine PD-L1 gene, and the construction of the pGEX-QPD-L1 recombinant expression plasmid;

[0009] S2, transformation expression and purification to obtain purified pGEX-QPD-L1 protein;

[0010] S3, Immunization of animals: Purified pGEX-QPD-L1 protein was used as an antigen to immunize mice.

[0011] S4, cell fusion, cell fusion of spleen cells and SP2 / 0 cells in immunized mice;

[0012] S5, screening of hybridoma cells yielded a stable hybridoma cell line 3B4C7B2F10C10 that secretes canine PD-L1 monoclonal antibody.

[0013] Further, the cloning of the canine PD-L1 gene, the cDNA reverse transcribed from the total RNA extracted from the peripheral blood of the canine is used as a template, and a primer is designed to perform PCR amplification to obtain the canine PD-L1 gene.

[0014] Further, the screening of the hybridoma cell is that after the positive hybridoma cell is subcloned and screened for four times in succession, a hybridoma cell strain 3B4C7B2F10C10 stably secreting the canine PD-L1 monoclonal antibody is obtained.

[0015] A monoclonal antibody against canine PD-L1 is secreted by the hybridoma cell strain.

[0016] As an improvement, the monoclonal antibody against canine PD-L1 comprises a light chain constant region and a heavy chain constant region, a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4.

[0017] As an improvement, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO. 1, and the nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO. 2.

[0018] The hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 or the monoclonal antibody against canine PD-L1 is applied to a biological preparation or a kit for detecting the PD-L1 protein of the canine tumor.

[0019] As an improvement, the kit is an immunodetection-based kit.

[0020] A kit for detecting the PD-L1 of the canine tumor comprises the hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 or the monoclonal antibody against canine PD-L1.

[0021] The hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 or the monoclonal antibody against canine PD-L1 is applied to the preparation of a reagent for assisting the diagnosis of the canine tumor.

[0022] As an improvement, the tumor is not limited to perianal epithelioma, melanoma, skin squamous carcinoma, hair acanthoma, breast cancer, lymphoma, osteosarcoma, prostate cancer, ovarian cancer, lung cancer.

[0023] Beneficial effects

[0024] Compared with the prior art, the canine PD-L1 monoclonal antibody, the hybridoma cell strain secreting the canine PD-L1 monoclonal antibody and the application thereof have the following advantages:

[0025] 1. The hybridoma cell strain secreting the anti-dog PD-L1 monoclonal antibody screened in the application can stably secrete the antibody, and after four times of subcloning screening and multiple identifications, it is ensured to be a monoclonal positive cell.

[0026] 2. The hybridoma cell strain screened in the application can secrete the dog PD-L1 monoclonal antibody, the antibody can specifically recognize the PD-L1 protein of the dog, color development is good in the immunohistochemical staining of the dog tumor, the degree of PD-L1 expression in the dog tumor can be accurately detected, the expression analysis of PD-L1 has important significance for the clinical application of the PD-1 / PD-L1 inhibitor in the dog, the high expression of PD-L1 can be used as an important marker of the immune therapy effect of the dog, the titer is high, specifically the ascites titer is 1:819200, the purified antibody titer is 1:51200, and the concentration of the purified antibody is 0.84 mg / ml; the specificity is strong, the dog PD-L1 protein can be specifically recognized in the immunohistochemical staining, which lays a foundation for the preparation of a commercial kit for the immunohistochemical detection of the dog tumor tissue and a dog tumor treatment drug based on the dog PD-L1 monoclonal antibody, supports the expression level of PD-L1 as an important reference factor for the future clinical trial design and treatment decision, and provides a reference for the immunohistochemical auxiliary diagnosis of the malignant degree of the dog tumor and the prognosis of the dog tumor immunotherapy mediated by the dog PD-1 and PD-L1 monoclonal antibodies, and has great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings are for the purpose of illustrating preferred embodiments and are not to be considered as limiting the application.

[0028] Figure 1 It is the nucleotide sequence alignment result chart of the dog PD-L1 in the embodiment of the application.

[0029] Figure 2 It is the PCR amplification chart of the dog PD-L1 gene in the embodiment of the application.

[0030] Figure 3 It is the double enzyme digestion identification chart of the dog pGEX-QPD-L1 recombinant vector in the embodiment of the application.

[0031] Figure 4 It is the Western-blot identification chart of the dog PD-L1 expression in the embodiment of the application.

[0032] Figure 5 It is the antibody purification chart of 3B4C7B2F10C10 in the embodiment of the application.

[0033] Figure 6 It is the chart of the dog PD-L1 monoclonal antibody recognizing pGEX-QPD-L1 in the embodiment of the application.

[0034] Figure 7Figure 1 shows the recognition of canine tumor tissues by the canine PD-L1 monoclonal antibody provided in the embodiments of the present application; wherein (a) is HE staining of canine perianal epithelioma (400x), (b) is IHC of canine perianal epithelioma (400x), (c) is HE of canine melanoma (400x), (d) is IHC of canine melanoma (400x), (e) is HE of canine skin squamous carcinoma (100x), and (f) is IHC of canine skin squamous carcinoma (400x). DETAILED DESCRIPTION

[0035] In order to better understand the above technical solutions, the technical solutions of the embodiments of the present application will be described in detail below with the aid of the accompanying drawings and specific embodiments. It should be understood that the specific features in the embodiments of the present application and the specific embodiments are detailed descriptions of the technical solutions of the embodiments of the present application, and are not limitations of the technical solutions of the present application. In the case of no conflict, the technical features in the embodiments of the present application and the specific embodiments can be combined with each other.

[0036] A hybridoma cell line secreting an anti-canine PD-L1 monoclonal antibody, the hybridoma cell line is 3B4C7B2F10C10, which was preserved in the China Center for Type Culture Collection on June 24, 2025, with the preservation number CCTCC NO: C2025167, and the address is Wuhan, China, Wuhan University.

[0037] As an improvement, the preparation method of the hybridoma cell line 3B4C7B2F10C10 comprises the following steps:

[0038] S1, cloning of canine PD-L1 gene, construction of pGEX-QPD-L1 recombinant expression plasmid;

[0039] S2, transformation, expression and purification, to obtain purified pGEX-QPD-L1 protein;

[0040] S3, immunizing animals, using the purified pGEX-QPD-L1 protein as an antigen to immunize mice;

[0041] S4, cell fusion, cell fusion of the spleen cells of the immunized mice with SP2 / 0 cells;

[0042] S5, screening of hybridoma cells, to obtain a hybridoma cell line 3B4C7B2F10C10 stably secreting canine PD-L1 monoclonal antibody.

[0043] Further, the cloning of the canine PD-L1 gene, the cDNA reverse transcribed from the total RNA extracted from the peripheral blood of the dog is used as a template, and primers are designed for PCR amplification to obtain the canine PD-L1 gene. It should be noted that PD-L1 is expressed in lymphocytes, and the peripheral blood of the dog contains lymphocytes, therefore, in the experiment, the total RNA is extracted directly from the peripheral blood, without first separating the lymphocytes and then extracting the RNA, which simplifies the experimental steps and saves time and resources.

[0044] Further, the screening of the hybridoma cells is that after four times of subcloning and screening of the positive hybridoma cells, a hybridoma cell strain 3B4C7B2F10C10 stably secreting the canine PD-L1 monoclonal antibody is obtained.

[0045] A monoclonal antibody against canine PD-L1 is secreted by the hybridoma cell strain described above, specifically obtained by immunizing mice with canine PD-L1 as an immunogen, and then screening after cell fusion and four times of subcloning. Immunohistochemical experiments show that the monoclonal antibody against canine PD-L1 has a strong positive reaction with canine malignant perianal epithelial tumors.

[0046] As an improvement, the monoclonal antibody against canine PD-L1 includes a light chain constant region and a heavy chain constant region, a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

[0047] As an improvement, the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO. 1, and the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO. 2.

[0048] The hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 described above or the monoclonal antibody against canine PD-L1 described above is used in a biological preparation or kit for detecting canine tumor PD-L1 protein.

[0049] As an improvement, the kit is an immunodetection-based kit.

[0050] A kit for detecting canine tumor PD-L1 includes the hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 described above or the monoclonal antibody against canine PD-L1 described above.

[0051] The hybridoma cell strain secreting the monoclonal antibody against canine PD-L1 described above or the monoclonal antibody against canine PD-L1 described above is used in the preparation of a reagent for assisting in the diagnosis of canine tumors.

[0052] As an improvement, the tumor is not limited to perianal epithelioma, melanoma, skin squamous carcinoma, hair acanthoma, breast cancer, lymphoma, osteosarcoma, prostate cancer, ovarian cancer, lung cancer.

[0053] Example 1 canine PD-L1 monoclonal antibody, hybridoma cell line secreting canine PD-L1 monoclonal antibody

[0054] I. Cloning of canine PD-L1 gene and construction of pGEX-QPD-L1 recombinant expression plasmid

[0055] (1) Canine PD-L1 amplification

[0056] First, according to the manufacturer's instructions, total RNA was extracted from canine peripheral blood using RNAprep Pure High-Efficiency Total RNA Extraction Kit (TIANGEN), and then reverse transcription was performed using PrimeScript II TM 1st Strand cDNASynthesis Kit (Takara) according to the published canine PD-L1 mRNA gene sequence (Gene Sequence No.: NM_001291972.1), the amplification primers were designed, and EcoRl and Xhol enzyme digestion sites were introduced upstream and downstream of the primers, and PhantaMax Super-Fidelity DNA Polymerase Kit (Novozyme) was used to amplify the target fragment, as shown in Figure 2 It can be seen that the amplified gene and the gene sequence on the database are 100% matched, and there is no amino acid mutation, which proves that the amplified gene is completely correct.

[0057] The amplification primers are shown in the following table:

[0058]

[0059] Template RNA denaturation and reverse transcription reaction:

[0060]

[0061] Denaturation and annealing reactions were performed on the PCR instrument as follows:

[0062] 65℃ 5min

[0063] 4℃

[0064] Reverse transcription reaction was performed on the PCR instrument as follows:

[0065] 42℃ 45min

[0066] 95℃ 5min

[0067] 4℃.

[0068] Purpose fragment amplification system:

[0069]

[0070] Purpose fragment amplification procedure:

[0071]

[0072] (2) PCR product and vector digestion (Takara)

[0073]

[0074]

[0075] 37℃, 15min.

[0076]

[0077] 37℃, 15min.

[0078] (3) QPD-L1 and pGEX-6p-1 connection (Takara)

[0079]

[0080] 25℃, 30min.

[0081] The recombinant plasmid was transformed into DH5α; 37℃ for 16h, the single colony was picked on the plate and shaken, the plasmid was extracted, and EcoR I and Xho I double enzyme digestion identification was performed, as shown in Figure 3 , the positive recombinant plasmid was named pGEX-QPD-L1, and was sent to Anshengda Company for sequencing, and the sequencing results were compared with NM_001291972.1 by Blast, and the results were 100% identical, as shown in Figure 1 .

[0082] II. Transformation, expression and purification

[0083] The correct pGEX-QPD-L1 recombinant plasmid and pGEX-6P-1 empty plasmid were transformed into BL21 (DE3) E. coli competent cells for induction expression, including:

[0084] (1) A single colony was picked from the LB plate with a final concentration of 100 μg / ml ampicillin antibiotic, and 2 mL of LB liquid medium with a final concentration of 100 μg / ml ampicillin antibiotic was inoculated, 37℃, 200 rpm, shaking for 16h.

[0085] (2) Take 200 μL of the bacteria liquid in the first step, add to 20 mL of LB liquid medium with a final concentration of 100 μg / ml ampicillin antibiotic.

[0086] (3) The bacteria solution in the second step was shaken for about 4 hours, and OD was measured 600 Close to 0.5-0.7, 20 mL of the bacteria solution was divided into two tubes, 10 mL in each tube, and 1 tube was added with IPTG at a final concentration of 1 mmol / L, and the other tube was not added with IPTG as a non-induced control, and the bacteria were shaken at 16°C and 180 rpm for 20 hours.

[0087] (4) The bacteria solution in the two tubes in the third step was centrifuged at 5000 rpm and 4°C for 5 minutes, the supernatant was discarded, 2 mL of PBS was added for suspension, and ultrasonic crushing was performed for 5 minutes (2 seconds on and 3 seconds off). The supernatant was retained after centrifugation at 12000 rpm and 4°C for 20 minutes, the precipitate was suspended with 2 mL of PBS, and Western-blot was performed to analyze whether the protein was expressed in a soluble form or in an inclusion body form. The results are shown in Figure 4 The protein was expressed in a large amount in a soluble form in the supernatant.

[0088] The correct pGEX-QPD-L1 recombinant plasmid was induced for large-scale expression and purification, including:

[0089] (1) A single colony was picked from an LB plate with an ampicillin antibiotic at a final concentration of 100 μg / ml, and 5 mL of LB liquid medium with an ampicillin antibiotic at a final concentration of 100 μg / ml was inoculated, and the bacteria were shaken at 37°C and 200 rpm for 16 hours.

[0090] (2) 4 mL of the bacteria solution in the first step was added to 400 mL of LB liquid medium with an ampicillin antibiotic at a final concentration of 100 μg / ml.

[0091] (3) The bacteria solution in the second step was shaken for about 4 hours, and OD was measured 600 Close to 0.5-0.7, 20 mL of the bacteria solution was divided into two tubes, 10 mL in each tube, and 1 tube was added with IPTG at a final concentration of 1 mmol / L, and the other tube was not added with IPTG as a non-induced control, and the bacteria were shaken at 16°C and 180 rpm for 20 hours.

[0092] (4) The bacteria solution in the third step was centrifuged at 5000 rpm and 4°C for 5 minutes, the supernatant was discarded, 8 mL of non-denaturing lysis solution was added for suspension, and the subsequent steps were performed according to the operation manual of the His-tag protein purification kit of the Biyun Tian company for protein purification. The concentration of the purified pGEX-QPD-L1 protein was 0.95 mg / mL.

[0093] III. Immunization of animals

[0094] Mouse immunization: 6-8 weeks old female BALB / c mice were used for immunization. The purified pGEX-QPD-Ll protein was mixed with Freund's complete adjuvant (Sigma) in equal volume and emulsified thoroughly, and then injected intraperitoneally at a dose of 100 μg per BALB / c mouse. Two weeks later, the mice were immunized for the second time, but Freund's incomplete adjuvant (Sigma) was used instead of Freund's complete adjuvant. Two weeks later, the mice were immunized for the third time, which was the same as the second time. Two weeks later, the mice were boosted, but no adjuvant was used, and the antigen was used at a dose of 200 μg. Three days later, the mice were used for cell fusion.

[0095] Four, cell fusion

[0096] The spleen cells of the immunized mice were fused with SP2 / 0 cells (commercially available). The fused cells were mixed with feeder cells (obtained from the peritoneal cavity of another non-immunized BALB / c mouse, which is a routine operation in the art), and then divided into 5 96-well cell culture plates. The cells were selected and cultured with HAT medium. About 10 days after the fusion, the cell supernatant was taken for screening.

[0097] Five, screening and expansion of hybridoma cells

[0098] Establishment of screening ELISA method: The matrix method was used to determine the coating antigen concentration, enzyme-labeled antibody dilution ratio, and positive and negative serum dilution multiples. The pGEX-QPD-Ll was used as the coating antigen, and was diluted by 2-fold from 1:100 to 1:12800. The positive serum was diluted by 2-fold from 1:100 to 1:204800. The negative serum was diluted by 2-fold from 1:100 to 1:6400. The HRP-labeled goat anti-mouse enzyme-labeled antibody was diluted by 2-fold from 1:5000 to 1:20000. The results showed that the optimal indirect ELISA conditions for screening were as follows: the coating antigen was diluted at 1:1600, the positive and negative sera were diluted at 1:1600 and 1:800, respectively, and the HRP-labeled goat anti-mouse enzyme-labeled antibody was diluted at 1:10000.

[0099] Screening of hybridoma cells:

[0100] When the cell supernatant turned yellow, the liquid was discarded, and the liquid was replenished. After 12 hours, the established indirect ELISA method was used for detection. The positive hybridoma cells were expanded into 24-well plates, and when they were fully grown, they were subjected to ELISA detection again. The positive cells were then subcloned. To ensure that a single hybridoma cell that stably secretes antibodies can be screened, the subcloning screening was repeated four times. A cell strain that stably secretes antibodies was obtained, and was named 3B4C7B2F10C10.

[0101] Expansion and cryopreservation of hybridoma cells:

[0102] The positive monoclonal hybridoma cells selected after four subcloning were successively expanded in culture in the order of 96-well plate, 24-well plate, 6-well plate, and T25 cell culture flask. When the cells were fully grown in each expansion, the established indirect ELISA method was used for detection, and the selected monoclonal cells were continuously passaged for 50 times to ensure stable secretion of antibodies. The positive monoclonal hybridoma cells in logarithmic growth phase, in good condition and meeting the requirements for cell number for freezing were frozen in liquid nitrogen for standby.

[0103] The hybridoma cell line is 3B4C7B2F10C10 (Hybridoma cell line 3B4C7B2F10C10), which was preserved in China Center for Type Culture Collection on June 24, 2025, with the preservation number CCTCC NO: C2025167 and the address Wuhan, China, Wuhan University.

[0104] Six, monoclonal antibody subclass identification, ascites preparation and antibody purification, monoclonal antibody ELISA titer, variable region sequence

[0105] Monoclonal antibody subclass identification:

[0106] The mouse monoclonal Ig class / subclass identification with HRP-labeled goat anti-mouse IgG was performed according to the instructions of the enzyme-labeled secondary antibody ready-to-use kit (Biolegend), and the subclass of 3B4C7B2F10C10 was IgG1.

[0107] Ascites preparation and antibody purification:

[0108] Select the experienced BALB / c mice, intraperitoneal injection of 500ul liquid paraffin, a week later, inoculate 3B4C7B2F10C10 hybridoma cells, each mouse inoculation 500-100 million hybridoma cells. A week or so, the mouse abdomen significantly swollen, can be used with 7 needle abdominal fluid. The specific steps are: ① 25mL ascites 12000rpm centrifugation 15min, take the supernatant in a small beaker, built-in magnetic stirrer on the magnetic bar light stirring, to avoid the production of bubbles, dropwise saturated ammonium sulfate 12.5mL, add time should be slow, light stirring 30min, 12000rpm centrifugation 15min. ② discard the supernatant, the precipitate was suspended with 33.3% saturated ammonium sulfate, 12000rpm centrifugation 15min, repeat step ② 2 times, the precipitate was dissolved in 3.75mL PBS. ③ dialysis bag with ultrapure water 10min, then use DDW dialysis bag. ④ the dialysis bag one end with clip, by the opening end of the crude antibody solution. ⑤ the dialysis bag is placed in 10 times the volume of the sample liquid volume of PBS, 4℃ dialysis 12h, change once PBS, continue 4℃ dialysis 12h. ⑥ collection of liquid, 12000rpm centrifugation 2min, take the supernatant, ProteinA280 measure the concentration of antibody protein. Crude antibody with PBS dilution 2 times, using ProteinA+G Agarose (Bi Yun Tian) purified antibody, the specific steps refer to the instructions, purified antibody SDS-PAGE electrophoresis identification, results as shown in Figure 5

[0109] Monoclonal antibody ELISA titer:

[0110] The established ELISA method was used to determine the hybridoma cell supernatant titer, the cell supernatant titer was 1:1600. The ascites titer was 1:819200, and the purified antibody titer was 1:51200.

[0111] Monoclonal antibody variable region sequence:

[0112] The 3B4C7B2F10C10 hybridoma cells in the 6 well plate were removed from the culture medium, washed with PBS for 2 times, and then 1mL Trizol was added to each well to lyse the cells. The cell lysate was collected in a 1.5mL centrifuge tube and stored at -80℃. Then the sample was submitted to a biotechnology company for sequencing of mouse hybridoma monoclonal antibody variable region. The sequencing results are as follows:

[0113] Amino acid sequence CDR1 CDR2 CDR3 Light chain variable region QSLLHSNGNTN KVS SQSTHVPWT Heavy chain variable region GYTFRNYG INTYTGEA SNMDY

[0114] ​Nucleic acid sequence of the heavy chain variable region: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCAGAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGATTTAAAGTTTATGGGCTGGATAAACACCTACACTGGAGAGGCAAAATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTTCAAATATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO. 2)

[0115] Nucleic acid sequence of the heavy chain variable region: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCAGAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGATTTAAAGTTTATGGGCTGGATAAACACCTACACTGGAGAGGCAAAATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTTCAAATATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO. 2)

[0116] Amino acid sequence of the heavy chain variable region: DVVLTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTNLHWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGRGSGTDFTLKISRVETEDLGVYFCSQSTHVPWTF GGGTKLEIK (SEQ ID NO. 3)

[0117] Amino acid sequence of heavy chain variable region: QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQ APGKDLKFMGWINTYTGEAKYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSNMD YWGQGTSVTVSS (SEQ ID NO. 4)

[0118] Example 2 Application of canine PD-L1 monoclonal antibody - recognizing canine PD-L1

[0119] The purified pGEX-QPD-L1 protein and pGEX-6P-1 empty vector protein were mixed with 6x protein loading buffer (Bi Yun Tian) respectively, heated at 100°C for 8 min, separated by 12% SDS-PAGE electrophoresis, then wet transferred to NC membrane at 400mA current for 35 min, the membrane was blocked with 5% skim milk powder PBS at room temperature for 2h, washed with PBST for 2 times, then added 1:100 diluted 3B4C7B2F10C10 cell supernatant, incubated at room temperature for 1h, washed with PBST for 3 times, then added 1:10000 diluted HRP labeled goat anti-mouse IgG secondary antibody (MBL), incubated at room temperature for 1h, washed with PBST for 3 times. Then developed, the results showed that the monoclonal antibody only recognized pGEX-QPD-L1 protein but not pGEX-6P-1 empty vector protein, and the Western-blot results are shown in Figure 6 .

[0120] Example 3 Canine PD-L1 monoclonal antibody recognizes canine perianal epithelioma, canine melanoma, canine skin squamous carcinoma.

[0121] The monoclonal antibody recognizes canine tumor tissues, specifically including:

[0122] (1) De-waxing: immerse the sections in xylene (I) and xylene (II) respectively for 15 min each on the staining rack for de-waxing.

[0123] (2) De-xylene: 100%, 95%, 85%, 75% concentration of ethanol de-xylene for 1 min each. Rinse with water for 30s.

[0124] (3) Inactivate endogenous peroxide: soak the slide in 1% hydrogen peroxide methanol for 30 min, wash with PBS for 3 times.

[0125] (4) Expose antigen: add 0.1% trypsin dropwise on the tissue, act at room temperature for 5 min, wash with PBS for 3 times.

[0126] (5) Blocking: add 5% BSA dropwise in the dark, cover the entire tissue, act at room temperature for 30 min.

[0127] (6) Incubate the first antibody: pour 5% BSA, dilute the monoclonal antibody with the antibody diluent (working concentration 1:500), incubate at 37°C for 1 h, and wash with PBS for 3 times.

[0128] (7) Incubate the second antibody: dilute the HRP-labeled goat anti-mouse IgG with the antibody diluent (working concentration 1:500), incubate at 37°C for 1 h, and wash with PBS for 3 times.

[0129] (8) DAB staining: add the DAB staining solution under light protection, observe the tissue staining at all times, and stain for 3 min, and terminate with DDW.

[0130] (9) Counterstaining: counterstain with hematoxylin for 1 min, and wash with PBS for 3 times.

[0131] (10) Differentiate: differentiate with 1% hydrochloric acid ethanol for 3 s, and quickly put into running water until the slice turns blue.

[0132] (11) Slice dehydration: dehydrate with 75%, 85%, 95%, and 100% ethanol for 10 s each.

[0133] (12) Slice deethanolization: immerse the slice in xylene (I) and xylene (II) for 5 min each to remove ethanol.

[0134] (13) Mounting: mount with neutral resin.

[0135] The immunohistochemical results are shown in Figure 7 . As can be seen from the figure, the monoclonal antibody secreted by the hybridoma cell 3B4C7B2F10C10 has strong binding with the tumor cells (the more brownish color, the more tumor cells), which can reflect the expression amount of canine PD-L1 in the tumor cells and the number of tumor cells in the tissue, further assisting in diagnosing the malignant degree of the tumor tissue and indicating the development degree of the tumor disease.

Claims

1. A hybridoma cell line secreting an anti-canine PD-Ll monoclonal antibody, characterized in that, The hybridoma cell line is 3B4C7B2F10C10 (Hybridoma cell line 3B4C7B2F10C10), which was deposited with the China Center for Type Culture Collection on June 24, 2025, and has the accession number CCTCC NO: C2025167, and is located at Wuhan University, Wuhan, China.

2. An anti-canine PD-Ll monoclonal antibody, characterized in that, The anti-dog PD-L1 monoclonal antibody is secreted by the hybridoma cell line of claim 1. 3.The anti-dog PD-L1 monoclonal antibody of claim 2, characterized in that, The anti-dog PD-L1 monoclonal antibody comprises a light chain constant region and a heavy chain constant region, a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4. 4.The anti-dog PD-L1 monoclonal antibody of claim 2, characterized in that, The nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO. 1, and the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.

2.

5. The hybridoma cell line secreting the anti-dog PD-L1 monoclonal antibody of claim 1 or the anti-dog PD-L1 monoclonal antibody of any one of claims 2-4 for use in a biological preparation or kit for detecting PD-L1 protein in a dog tumor.

6. Use according to claim 5, characterized in that, The kit is an immunodetection-based kit.

7. A kit for detecting PD-L1 in a canine tumor, the kit comprising: a) a first antibody that specifically binds to PD-L1; and b) a second antibody that specifically binds to the first antibody. The kit comprises the hybridoma cell line secreting the anti-dog PD-L1 monoclonal antibody of claim 1 or the anti-dog PD-L1 monoclonal antibody of any one of claims 2-4.

8. The hybridoma cell line secreting the anti-dog PD-L1 monoclonal antibody of claim 1 or the anti-dog PD-L1 monoclonal antibody of any one of claims 2-4 for use in the preparation of a reagent for the auxiliary diagnosis of a dog tumor.

9. Use according to claim 8, characterized in that, The tumor includes, but is not limited to, perianal epithelioma, melanoma, cutaneous squamous carcinoma, hair acanthoma, breast cancer, lymphoma, osteosarcoma, prostate cancer, ovarian cancer, lung cancer.

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