Cat PD-L1 monoclonal antibody, hybridoma cell strain secreting cat PD-L1 monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a cat PD-L1 monoclonal antibody, a hybridoma cell strain secreting the cat PD-L1 monoclonal antibody and application of the hybridoma cell strain, and relates to the technical field of immunology and genetic engineering antibodies. The cat PD-L1 monoclonal antibody is secreted by a hybridoma cell strain YMPDL-1, the hybridoma cell strain YMPDL-1 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2024251, and the preservation date is August 31, 2024. The cat PD-L1 monoclonal antibody provided by the invention can specifically recognize dog and cat PD-L1 proteins, can further analyze dog and cat tumor diseases, can also provide a necessary tool for related scientific research, and has a huge application prospect.

Description

Technical Field

[0001] The present application relates to the field of immunology and genetic engineering antibody technology, and in particular to feline PD-L1 monoclonal antibodies, hybridoma cell lines secreting feline PD-L1 monoclonal antibodies, and applications thereof. Background Art

[0002] Common tumor diseases in dogs and cats include breast cancer, lymphoma, subcutaneous squamous cell carcinoma, etc. The diagnosis of tumor diseases is mainly through clinical examination and imaging, which is difficult to confirm. In recent years, in medicine, immunohistochemical diagnosis technology based on marker molecule monoclonal antibodies has been widely used around the world. Programmed death ligand-1 (PD-L1) is expressed in large quantities in tumor cells and epithelial cells. It is an important tumor surface marker molecule and is the most widely used. However, there is no research and application in this area in veterinary medicine. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0004] In a first aspect of the present invention, a feline PD-L1 monoclonal antibody is provided, wherein the feline PD-L1 monoclonal antibody is secreted by a hybridoma cell line YMPDL-1, and the hybridoma cell line YMPDL-1 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2024251, a deposit date of August 31, 2024, and an address of the depository unit of Wuhan University, Wuhan, China, and is classified and named as Hybridoma cell line YMPDL-1.

[0005] Furthermore, the preparation method of the hybridoma cell line YMPDL-1 comprises:

[0006] Cloning of feline PD-L1 gene and construction of PET-MPD-L1 recombinant expression plasmid;

[0007] Transformation, expression and purification to obtain purified PET-MPD-L1 protein;

[0008] Immunizing animals: using purified PET-MPD-L1 protein as an antigen to immunize mice;

[0009] Cell fusion: spleen cells from immunized mice were fused with SP2 / 0 cells;

[0010] Through the screening of hybridoma cells, a hybridoma cell line YMPDL-1 that stably secretes feline PD-L1 monoclonal antibody was obtained.

[0011] Furthermore, the cloning of the cat PD-L1 gene includes: using the cDNA extracted from the total RNA extracted from the cat's peripheral blood and reverse transcribed therefrom as a template, designing primers for PCR amplification, and obtaining the cat PD-L1 gene.

[0012] Furthermore, the screening of hybridoma cells includes: after three consecutive subcloning and screening of positive hybridoma cells, a hybridoma cell line YMPDL-1 that stably secretes feline PD-L1 monoclonal antibodies is obtained.

[0013] The second aspect of the present invention provides a hybridoma cell line, wherein the hybridoma cell line YMPDL-1 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2024251 and a deposit date of August 31, 2024. The depository address is Wuhan University, Wuhan, China, and the classification name is Hybridoma cell line YMPDL-1.

[0014] In a third aspect of the present invention, there is provided an application of the cat PD-L1 monoclonal antibody, wherein the cat PD-L1 monoclonal antibody is used to prepare a biological agent for recognizing the PD-L1 protein in cat tumor tissue, and / or the cat PD-L1 monoclonal antibody is used to prepare a biological agent for recognizing the PD-L1 protein in dog tumor tissue.

[0015] The application of the cat PD-L1 monoclonal antibody, the application of the cat PD-L1 monoclonal antibody in the preparation of a biological preparation that identifies the cat PD-L1 protein, and / or the application of the cat PD-L1 monoclonal antibody in the preparation of a biological preparation that identifies the canine PD-L1 protein.

[0016] The application of the cat PD-L1 monoclonal antibody, the application of the cat PD-L1 monoclonal antibody in the preparation of an immunosuppressant for treating cat tumors, and / or the application of the cat PD-L1 monoclonal antibody in the preparation of an immunosuppressant for treating canine tumors.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] The cat PD-L1 monoclonal antibody provided by the present invention can specifically recognize the cat PD-L1 protein, and can also specifically recognize the canine PD-L1 protein, and can further analyze canine and cat tumor diseases, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application.

[0020] Figure 1 This is a diagram of the cat PD-L1 nucleotide sequence alignment results provided in the examples of this application.

[0021] Figure 2 This is a comparison diagram of the canine and feline PD-L1 structure modeling provided in the examples of the present application.

[0022] Figure 3 This is a PCR amplification diagram of the cat PD-L1 gene provided in the examples of the present application.

[0023] Figure 4 This is a double enzyme digestion identification diagram of the PET-MPD-L1 recombinant vector provided in the examples of this application.

[0024] Figure 5 This is a Western-blot identification diagram of cat PD-L1 expression provided in the examples of the present application, M: protein molecular mass standard; 1: PET-32a empty vector supernatant protein; 8: PET-MPD-L1 supernatant protein.

[0025] Figure 6 This is a purification diagram of cat PD-L1 provided in the examples of this application. BL21 (PET-MPD-L1) was induced by IPTG and then purified by Ni 2+ Affinity chromatography purification was performed and PET-MPD-L1 SDS-PAGE electrophoresis was performed, wherein M: protein molecular weight standard; 5-7: protein washing solution; 8-10: protein eluent.

[0026] Figure 7 This is a diagram of the YMPDL-1 antibody purification provided in the examples of the present application.

[0027] Figure 8 This is a PET-MPD-L1 diagram of cat PD-L1 monoclonal antibody recognition provided in the examples of the present application.

[0028] FIG9 is a diagram of canine tumor tissue recognition by cat PD-L1 monoclonal antibody provided in the embodiment of the present application; wherein, Figure 9a HE staining of canine perianal epithelial tumor (100×). Figure 9b This is a canine perianal epithelial tumor IHC (400×). Fig.9c This is a canine benign tricholoma (a small number of positive cells) IHC (400×). Figure 9d This is a canine benign mammary tumor (positive) IHC (400×), Fig.9e Canine malignant mammary tumor (strongly positive) IHC (400×), Figure 9f This is canine adjacent cancer tissue IHC (400×).

[0029] FIG10 is a diagram showing the recognition of malignant lymphoma tissue of orange cats by the cat PD-L1 monoclonal antibody provided in the embodiment of the present application; wherein, Fig.10a HE staining of malignant lymphoma of orange cat (400×). Fig.10b This is IHC of malignant lymphoma in a orange cat (400×). DETAILED DESCRIPTION

[0030] In order to better understand the above-mentioned technical scheme, the technical scheme of the embodiments of the present application is described in detail below through the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical scheme of the embodiments of the present application, rather than limitations on the technical scheme of the present application. In the absence of conflict, the embodiments of the present application and the technical features in the embodiments may be combined with each other.

[0031] In order to obtain a biological preparation for diagnosing neoplastic diseases in dogs and cats, the embodiment of the present invention involves expressing cat PD-L1 as an antigen to prepare a monoclonal antibody, and a hybridoma cell YMPDL-1 secreting cat PD-L1 monoclonal antibody against the protein is prepared. The hybridoma cell YMPDL-1 is deposited in the China Center for Type Culture Collection of Wuhan University, with a deposit number of CCTCCNO: C2024251, a deposit date of August 31, 2024, and a deposit address of Wuhan University, Wuhan, China. The classification name is hybridoma cell line YMPDL-1. The cat PD-L1 monoclonal antibody can recognize cat PD-L1 and can also recognize dog PD-L1, and has good color development in cat tumor immunohistochemistry and can also have good color development in canine tumor immunohistochemistry. The immunohistochemistry technology based on YMPDL-1 can diagnose cat tumors and can also diagnose canine tumors, and has a large application space.

[0032] Furthermore, the cat PD-L1 monoclonal antibody of the embodiment of the present invention can accurately detect the degree of PD-L1 expression in canine tumors, and can also accurately detect the degree of PD-L1 expression in cat tumors. The expression analysis of PD-L1 is of great significance to the clinical application of PD-1 / PD-L1 inhibitors in dogs and cats. High expression of PD-L1 can be used as an important marker for the immunotherapy effect of dogs and cats, and supports the use of PD-L1 expression level as an important reference factor for future clinical trial design and treatment decisions. Since there are very few monoclonal antibodies that react to canine and feline PD-L1 worldwide, there are no commercial anti-canine and feline PD-L1 monoclonal antibodies in China.

[0033] The present invention provides a feline PD-L1 monoclonal antibody, which is obtained by using feline PD-L1 as an immunogen, immunizing mice, and then undergoing cell fusion and three subclonings before screening. Since the homology between canine and feline PD-L1 structures is very high, the monoclonal antibody against feline PD-L1 can recognize canine PD-L1, and through immunohistochemical tests, it is found that the monoclonal antibody against feline PD-L1 has a strong positive reaction with canine malignant breast cancer.

[0034] Specifically, the hybridoma cell line YMPDL-1, the preparation method comprises:

[0035] Cloning of feline PD-L1 gene and construction of PET-MPD-L1 recombinant expression plasmid;

[0036] Transformation, expression and purification to obtain purified PET-MPD-L1 protein;

[0037] Immunizing animals: using purified PET-MPD-L1 protein as an antigen to immunize mice;

[0038] Cell fusion: spleen cells from immunized mice were fused with SP2 / 0 cells;

[0039] Through the screening of hybridoma cells, a hybridoma cell line YMPDL-1 that stably secretes feline PD-L1 monoclonal antibody was obtained.

[0040] Among them, the cloning of the cat PD-L1 gene was carried out by using the cDNA extracted from the total RNA reverse transcribed from the cat's peripheral blood as a template, designing primers for PCR amplification, and obtaining the cat PD-L1 gene. It is understandable that PD-L1 is expressed in lymphocytes, and cat peripheral blood contains lymphocytes. We directly extracted total RNA from peripheral blood without separating lymphocytes first and then extracting RNA, which simplified the experimental steps and saved time and resources.

[0041] In addition, after three consecutive subcloning and screening of positive hybridoma cells, a hybridoma cell line YMPDL-1 that stably secretes feline PD-L1 monoclonal antibodies was obtained. This is because in our experiments, we found that after the second subcloning, the subcloning process may cause poor growth or inability to continue to proliferate. After three subclonings, genetically stable hybridoma cells can be screened.

[0042] In the embodiments of the present invention, the cat PD-L1 monoclonal antibody can be used to identify the PD-L1 protein in cat and / or dog tumor tissues; it can also be used to identify the PD-L1 protein in other tissues of cats and / or dogs; it can also be used in the preparation of immunosuppressants for treating cat and / or dog tumors. Regarding the application of the cat PD-L1 monoclonal antibody, since high expression of PD-L1 can be used as an important marker for the immunotherapy effect of dogs or cats, and supports the use of PD-L1 expression level as an important reference factor for future clinical trial design and treatment decisions, the expression analysis of canine and cat PD-L1 in canine and cat tumors is of great significance to the clinical application of PD-1 / PD-L1 inhibitors in dogs and cats. The cat PD-L1 monoclonal antibody of the present invention can specifically identify canine PD-L1 and cat PD-L1, and can further analyze canine and cat tumor diseases, and has great application prospects.

[0043] Example 1 Feline PD-L1 monoclonal antibody, hybridoma cell line secreting feline PD-L1 monoclonal antibody 1. Cloning of feline PD-L1 gene, construction of PET-MPD-L1 recombinant expression plasmid

[0044] (1) Feline PD-L1 amplification

[0045] First, total RNA was extracted from cat peripheral blood using RNAprep Pure High-efficiency Blood Total RNA Extraction Kit (TIANGEN) according to the manufacturer's instructions, and then PrimeScript II TM 1st Strand cDNA Synthesis Kit (Takara) was used for reverse transcription. Amplification primers were designed based on the published cat PD-L1 mRNA gene sequence (gene sequence number: XM_006939039), and EcoRl and Xhol restriction sites were introduced upstream and downstream of the primers. The PhantaMax Super-Fidelity DNA Polymerase Kit (Novozyme) was used to amplify the target fragment. Figure 3 As shown, it can be seen that the amplified gene is 100% aligned with the gene sequence in the database, and there is no amino acid mutation, proving that the amplified gene is completely correct.

[0046] The amplification primers are shown in the following table:

[0047]

[0048] Template RNA denaturation and reverse transcription reaction:

[0049]

[0050] Perform denaturation and annealing reactions on a PCR instrument as follows:

[0051] 65℃ 5min

[0052] 4℃

[0053] Perform reverse transcription reaction on PCR instrument according to the following conditions 42℃ 30min

[0054] 95℃ 5min

[0055] 4℃.

[0056] Target fragment amplification system:

[0057]

[0058]

[0059]

[0060] Target fragment amplification procedure:

[0061]

[0062] (2) Enzyme digestion of PCR product and vector (Takara)

[0063]

[0064] 37℃, 3.5h.

[0065]

[0066] 37℃, 3.5h.

[0067] (3) Linking of MPD-L1 and PET-32a (Takara)

[0068]

[0069]

[0070] 25℃, 2h.

[0071] The recombinant plasmid was transformed into DH5α; incubated at 37℃ for 16h, a single colony was picked from the plate and shaken to extract the plasmid, and then double-digested with EcoRⅠ and XhoⅠ for identification. Figure 4 As shown, the positive recombinant plasmids were named pET-32a-MPD-L1 and sent to BGI for sequencing. The sequencing results were blasted with XM_006939039.3, and the results were 100% identical, as shown in Figure 1 shown.

[0072] 2. Transformation, expression and purification

[0073] Transform the correct PET-MPD-L1 recombinant plasmid and PET-32a empty plasmid into BL21(DE3) E. coli competent cells and induce expression, including:

[0074] (1) Pick a single colony from the ampicillin plate and inoculate 2 mL of ampicillin LB. Shake the plate at 37°C, 200 rpm, for 16 h.

[0075] (2) Take 200 μL of the bacterial solution from step 1 and add it to 20 mL of LB containing ampicillin.

[0076] (3) The bacterial solution in step 2 was shaken for about 3 h. When the OD600 was close to 0.5-0.7, 20 mL of the bacterial solution was divided into 2 tubes, each with 10 mL. One tube was added with IPTG at a final concentration of 1 mM, and the other tube was not added with IPTG as an uninduced control. The culture was shaken for 20 h at 16°C and 180 rpm.

[0077] (4) After the third step of induction, centrifuge the bacterial solution at 5000 rpm and 4 degrees for 5 minutes, discard the supernatant, add 2 mL PBS to suspend, and perform ultrasonic disruption, 2 seconds on, 3 seconds off, and 5 minutes of ultrasonic. Centrifuge at 4°C, 12000 rpm for 20 minutes, retain the supernatant, suspend the precipitate with 2 mL PBS, and perform SDS-PAGE to analyze whether the protein is expressed in soluble form or inclusion body. Figure 5 As shown, the protein was expressed in large amounts in the supernatant in a soluble form.

[0078] The correct PET-MPD-L1 recombinant plasmid was induced to express in large quantities and purified, including:

[0079] (1) Pick a single colony from the ampicillin plate and inoculate 5 mL of ampicillin LB. Shake the plate at 37°C, 200 rpm, for 16 h.

[0080] (2) Take 4 mL of the bacterial solution from step 1 and add it to 400 mL of LB containing ampicillin.

[0081] (3) The bacterial solution in step 2 was shaken for about 3 hours. When the OD600 was close to 0.5-0.7, IPTG was added to a final concentration of 1 mM. The mixture was shaken at 16°C, 180 rpm, and the mixture was shaken for 20 hours.

[0082] (4) After the induction in the third step, the bacterial solution was centrifuged at 5000 rpm and 4 degrees for 5 min, the supernatant was discarded, and 8 mL of non-denaturing lysis buffer was added for suspension. The subsequent steps were carried out according to the operating instructions of the His tag protein purification kit produced by Biyuntian Company. The purification results are shown in the figure. Figure 6 As shown, the protein concentration was 1.15 mg / mL.

[0083] 3. Immunization of Animals

[0084] Mouse immunization: Take 6-8 week old female BALB / c mice, for the first immunization, the purified PET-MPD-L1 protein was mixed with Freund's complete adjuvant (Sigma) in equal volumes and fully emulsified, and injected intraperitoneally, 100 μg per BALB / c mouse. Two weeks later, a second immunization was performed. Unlike the first immunization, the second immunization used Freund's incomplete adjuvant (Sigma). Two weeks later, a third immunization was performed, and the third immunization was the same as the second immunization. Two weeks later, a booster immunization was performed. The booster immunization did not require an adjuvant, but the antigen amount was 200 μg. Three days later, cell fusion was performed.

[0085] IV. Cell Fusion

[0086] The spleen cells of the immunized mice were fused with SP2 / 0 cells. The fused cells were mixed with feeder cells and then distributed in 6 96-well cell culture plates. The cells were cultured with HAT medium and the cell supernatant was screened about 10 days after fusion.

[0087] 5. Screening of hybridoma cells

[0088] Establishment of screening ELISA method: Matrix method was used to determine the coating antigen concentration, enzyme-labeled antibody dilution ratio, and positive and negative serum dilution multiples. PET-MPD-L1 was used as coating antigen and diluted 2-fold from 1:400 to 1:6400, positive serum was diluted 2-fold from 1:800 to 1:1638400, negative serum was diluted 2-fold from 1:50 to 1:102400, and HRP-labeled goat anti-mouse enzyme-labeled antibody was diluted 2-fold from 1:5000 to 1:20000. Results The most suitable indirect ELISA conditions for screening were coating antigen diluted 1:1600, negative and positive serum diluted 1:1600 and 1:51200 respectively, and HRP-labeled goat anti-mouse enzyme-labeled antibody diluted 1:10000.

[0089] Screening of hybridoma cells: When the cell supernatant turns yellow, the established indirect ELISA method is used for detection. Positive hybridoma cells are expanded into 24-well plates and subcloned when they are full. After three consecutive subclones and screening, a cell line that stably secretes antibodies is obtained, named YMPDL-1, which is expanded and frozen in liquid nitrogen.

[0090] VI. Monoclonal antibody subclass identification, ascites preparation and antibody purification, monoclonal antibody ELISA titer

[0091] Monoclonal antibody subclass identification: Ig class / subclass identification was performed using HRP-labeled goat anti-mouse IgG mouse monoclonal antibody according to the instructions of the enzyme-labeled secondary antibody ready-to-use kit (Biolon), and the subclass of YMPDL-1 was IgG 2a.

[0092] Ascites preparation and antibody purification: Select multiparous BALB / c female mice, inoculate 0.5mL liquid paraffin intraperitoneally, and one week later, inoculate YMPDL-1 hybridoma cells, with 500,000-1 million hybridoma cells inoculated per mouse. After about a week, the mouse abdomen is significantly enlarged, and the ascites can be collected with a No. 7 needle. ① Centrifuge 25mL of ascites at 12000rpm / min for 15min, take the supernatant in a small beaker, stir gently on a magnetic stirrer with a built-in magnetic bar to avoid bubbles, add 12.5mL of saturated ammonium sulfate, add slowly, stir gently for 30min, and centrifuge at 12000rpm / min for 15min. ② Discard the supernatant, suspend the precipitate with 33.3% saturated ammonium sulfate, centrifuge at 12000rpm / min for 15min, repeat step ② twice, and the last precipitate is dissolved in 3.75mL PBS. ③ Boil the dialysis bag with ultrapure water for 10min, and then wash the dialysis bag thoroughly with DDW. ④ Clamp one end of the dialysis bag with a clamp and add the crude antibody solution from the open end. ⑤ Place PBS with a volume 10 times the volume of the sample liquid in the dialysis bag, dialyze at 4°C for 12 hours, change PBS once, and continue to dialyze at 4°C for 12 hours. ⑥ Collect the liquid, centrifuge at 12000rpm / min for 2min, take the supernatant, and measure the antibody protein concentration by OD280. After the crude antibody is diluted 2 times with PBS, use Protein A+G Agarose pre-packed column (Biyuntian) to purify the antibody. Refer to the instructions for specific steps. The purified antibody is identified by SDS-PAGE electrophoresis. The purity is good. The antibody purification results are as follows: Figure 7 shown.

[0093] Monoclonal antibody ELISA titer: The established ELISA method was used to determine the titer of hybridoma cell supernatant, and the cell supernatant titer was 1:3200. The ascites titer was 1:102400, and the purified antibody titer was 1:3200.

[0094] Example 2 Application of feline PD-L1 monoclonal antibody - recognition of feline PD-L1

[0095] After the purified PET-MPD-L1 and PET-32a empty carrier proteins were mixed with 5× protein loading buffer (Biyuntian), heated at 100℃ for 5min, separated by 12% SDS-PAGE electrophoresis, and then semi-dry transferred to NC membrane with 90mA current for 30min. The membrane was blocked with 10% skim milk powder PBS at room temperature for 2h, washed twice with PBST, and then added with 1:100 diluted YMPDL-1 cell supernatant, incubated at room temperature for 1h, washed three times with PBST, and then added with 1:10000 diluted HRP-labeled goat anti-mouse IgG secondary antibody (MBL), incubated at room temperature for 1h, and washed three times with PBST. Then developed, the monoclonal antibody only recognized PET-MPD-L1 but not PET-32a empty carrier protein. The Western-blot results are as follows: Figure 8shown.

[0096] Example 3 Application of feline PD-L1 monoclonal antibody - recognition of canine PD-L1

[0097] To determine the homology differences between canine and feline PD-L1 structures, the canine and feline PD-L1 structures were analyzed.

[0098] First, the homology modeling method was used to predict the structure of canine and feline PD-L1. The AlphaFold DB model of the protein containing the Ig-like domain of cat PD-L1 A0A5N3WM06.1.A A0A5N3WM06_MUNMU was used as a template, and the AlphaFold DB model of programmed cell death 1 ligand 1 Q9NZQ7.1.A PD1L1_HUMAN of canine PD-L1 was used as a template. The SWISS-MODEL workspace (https: / / swissmodel.expasy.org / ) was used to predict the three-dimensional structure of canine and feline PD-L1. The three-dimensional structures of canine PD-L1 and cat PD-L1 were overlapped and displayed in green and blue respectively using PyMOL 2.6.0a0 software ( Figure 2 ), the results showed that the homology between the canine and feline PD-L1 structures is very high, so the monoclonal antibody that recognizes cat PD-L1 should also be able to recognize canine PD-L1. Therefore, an IHC experiment of YMPDL-1 recognizing canine tumor tissue was performed, and the results were consistent with expectations, recognizing PD-L1 in canine malignant breast cancer, showing a strong positive result.

[0099] Example 4 Cat PD-L1 monoclonal antibody recognizes cat tumor tissue and dog tumor tissue

[0100] Monoclonal antibodies recognize tumor tissues in dogs and cats, including:

[0101] (1) Dewaxing: Place the sections on a staining rack and immerse them in xylene (I) and xylene (II) for 15 min each for dewaxing.

[0102] (2) Xylene removal: Use 100%, 95%, 85%, and 75% ethanol to remove xylene for 1 min each. Rinse with water for 30 s.

[0103] (3) Inactivation of endogenous peroxides: Soak the slides in 1% hydrogen peroxide in methanol for 30 min and wash three times with PBS.

[0104] (4) Antigen exposure: 0.1% trypsin was added to the tissue and allowed to act at room temperature for 5 min. The tissue was then washed three times with PBS.

[0105] (5) Blocking: Add 5% BSA in a dark place to cover the entire tissue and leave it in the room temperature for 30 min.

[0106] (6) Incubation with primary antibody: Pour 5% BSA, dilute the monoclonal antibody with antibody diluent (working concentration 1:500), incubate at 37°C for 1 h, and wash three times with PBS.

[0107] (7) Incubation with secondary antibody: dilute HRP-labeled goat anti-mouse IgG (working concentration 1:500) with antibody diluent, incubate at 37°C for 1 h, and wash three times with PBS.

[0108] (8) DAB staining: Add DAB staining solution in a dark place and observe the tissue staining at all times. The staining time is 3 min and the staining is terminated with distilled water.

[0109] (9) Staining: Stain with hematoxylin for 1 min and wash three times with PBS.

[0110] (10) Differentiation: Use 1% hydrochloric acid ethanol to differentiate for 3 seconds, and then quickly rinse in clean running water until the sections turn blue.

[0111] (11) Dehydration of sections: dehydrate in 75%, 85%, 95%, and 100% ethanol for 10 seconds each.

[0112] (12) Deethanolization of sections: Immerse the sections in xylene (I) and xylene (II) for 5 min each to remove ethanol.

[0113] (13) Sealing: Use neutral resin to seal the slide.

[0114] The immunohistochemistry results are shown in Figures 9 and 10. It can be seen that the monoclonal antibody secreted by the hybridoma cell line YMPDL-1 has a strong binding affinity with tumor cells (the more brown, the more tumor cells), but does not bind to normal cells. This indicates that tumor cells and normal cells can be accurately distinguished, and the number of tumor cells in the tissue can be seen, indicating the degree of development of the tumor disease.

[0115] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed. The above are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application. The above are only preferred implementation methods of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present application, and these improvements and variations should also be regarded as the protection scope of the present application.

Claims

1. A feline PD-L1 monoclonal antibody, characterized in that The feline PD-L1 monoclonal antibody is secreted by the hybridoma cell line YMPDL-1, and the hybridoma cell line YMPDL-1 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCCNO: C2024251 and the deposit date August 31, 2024.

2. The cat PD-L1 monoclonal antibody according to claim 1, characterized in that The preparation method of the hybridoma cell line YMPDL-1 comprises: Cloning of feline PD-L1 gene and construction of PET-MPD-L1 recombinant expression plasmid; Transformation, expression and purification to obtain purified PET-MPD-L1 protein; Immunizing animals: using purified PET-MPD-L1 protein as an antigen to immunize mice; Cell fusion: spleen cells from immunized mice were fused with SP2 / 0 cells; Through the screening of hybridoma cells, a hybridoma cell line YMPDL-1 that stably secretes feline PD-L1 monoclonal antibody was obtained.

3. The feline PD-L1 monoclonal antibody according to claim 2, characterized in that The cloning of the cat PD-L1 gene includes: using the cDNA extracted from the total RNA extracted from the cat peripheral blood and reverse transcribed therefrom as a template, designing primers for PCR amplification, and obtaining the cat PD-L1 gene.

4. The feline PD-L1 monoclonal antibody according to claim 2, characterized in that The screening of hybridoma cells comprises: after three consecutive subcloning and screening of positive hybridoma cells, a hybridoma cell line YMPDL-1 which stably secretes feline PD-L1 monoclonal antibodies is obtained.

5. A hybridoma cell line, characterized in that: The hybridoma cell line YMPDL-1 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: C2024251 and the deposit date August 31, 2024.

6. The use of the feline PD-L1 monoclonal antibody according to any one of claims 1 to 4, characterized in that: The cat PD-L1 monoclonal antibody is used to prepare a biological preparation for recognizing the PD-L1 protein in cat tumor tissue, and / or, the cat PD-L1 monoclonal antibody is used to prepare a biological preparation for recognizing the PD-L1 protein in dog tumor tissue.

7. The use of the feline PD-L1 monoclonal antibody according to any one of claims 1 to 4, characterized in that: Use of the cat PD-L1 monoclonal antibody in the preparation of a biological preparation for identifying the cat PD-L1 protein, and / or use of the cat PD-L1 monoclonal antibody in the preparation of a biological preparation for identifying the canine PD-L1 protein.

8. The use of the feline PD-L1 monoclonal antibody according to any one of claims 1 to 4, characterized in that: Use of the cat PD-L1 monoclonal antibody in the preparation of an immunosuppressant for treating cat tumors, and / or use of the cat PD-L1 monoclonal antibody in the preparation of an immunosuppressant for treating canine tumors.

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