Hybridoma cell strain secreting anti-fish grizyme B monoclonal antibody and application thereof

Abstract

The invention discloses a hybridoma cell strain capable of secreting an anti-fish grizyme B monoclonal antibody and application of the hybridoma cell strain, and relates to the field of fish immunology and disease prevention and control. The hybridoma cell strain 2C8H3 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number of the hybridoma cell strain 2C8H3 is CCTCC NO: C2024141, and the preservation date of the hybridoma cell strain 2C8H3 is May 9, 2024. According to the invention, tilapia granzyme B protein is purified through an eukaryotic expression system and is used as an antigen to immunize a mouse, and a screening and identification method combining flow cytometry, semi-quantitative detection and enzyme-linked immunosorbent assay is constructed on the basis, so that the monoclonal antibody capable of specifically recognizing and resisting nile tilapia granzyme B molecules is successfully prepared; and an effective tool and technical support are provided for researching the adaptive immune mechanism of fish, especially researching cytotoxic T lymphocytes.

Description

Technical Field

[0001] The present invention belongs to the fields of fish immunology and disease prevention and control, and relates to a hybridoma cell line secreting anti-fish granzyme B monoclonal antibody, a preparation method thereof, a monoclonal antibody, and an application of the monoclonal antibody in fish adaptive immunity. Background Art

[0002] T cells are an important part of adaptive immunity and play important roles such as killing target cells and secreting cytokines. According to different surface marker molecules of T cells, T cells can be divided into CD4 + T cells and CD8 + T cell subsets. Among them, activated CD8 + T cells are called cytotoxic T cells (CTLs), which can secrete cytotoxic factors and lyse infected cells, playing a key role in resisting intracellular pathogens such as viruses. As a cytotoxic factor, the perforin-granzyme pathway is an important pathway for CTLs to exert their killing function. Granzyme B is the most potent pro-apoptotic granzyme, similar to cysteine proteases (caspases), and can cleave after the aspartic acid (Asp) residue of the substrate. In the former mode, granzyme B enters the target cell through the transmembrane channel formed by perforin A punching holes on the cell surface, activates cysteine aspartic acid proteases caspases (such as caspase-3 and caspase-7), and induces apoptosis of the target cell. In the latter mode, granzyme B hydrolyzes the Bid protein in the BH 3 subfamily in the pro-apoptotic family to indirectly initiate this pathway.

[0003] Compared with mammals, the research on fish adaptive cellular immunity is relatively scattered. Fish have a special position in the evolutionary history and are the earliest vertebrates with adaptive immunity. There are also various T cell subsets and granzyme B proteins in fish. However, at present, there is little research on the immune process of CD8 + T cells in teleost fish and on how granzyme B exerts its cytotoxic effect in CTLs. This may be due to certain difficulties in the process of purifying teleost fish granzyme B recombinant protein and preparing monoclonal antibodies, which has limited the development of fish T cell research to a certain extent.

[0004] It plays an irreplaceable role in adaptive immunity. However, due to the lack of monoclonal antibodies against T lymphocyte-related cytotoxic cytokines, issues such as the toxic function and mechanism of action of T lymphocytes cannot be studied in depth.

[0005] Therefore, the research and development of specific monoclonal antibodies against tilapia granzyme B can provide a powerful tool for studying the toxic function of tilapia T cells, and will provide technical support and theoretical basis for in-depth exploration of the adaptive immune response mechanism of tilapia and fish disease control. Summary of the Invention

[0006] In view of the existing technical problems in the research on fish cytotoxic T cell immunity, the present invention has developed a preparation and screening technology for monoclonal antibodies (mAbs) based on a eukaryotic expression system, and successfully prepared a hybridoma cell line 2C8H3 that can stably secrete mAbs against granzyme B of Nile tilapia (Oreochromis niloticus). The antibody secreted by it can specifically recognize and bind to tilapia lymphocytes expressing granzyme B molecules, providing an effective tool and technical support for exploring the adaptive immune mechanism of fish, especially the research on cytotoxic T lymphocytes.

[0007] The technical solutions provided by the present invention to solve the above technical problems are as follows:

[0008] In the first aspect, the present invention discloses a hybridoma cell line 2C8H3 (Hybridoma cell line 2C8H3) that secretes monoclonal antibodies against fish granzyme B. The hybridoma cell line 2C8H3 is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2024141, and the deposit date is May 9, 2024.

[0009] In the second aspect, the present invention discloses a preparation method of the hybridoma cell line 2C8H3 as described in the first aspect, including the following steps:

[0010] Construct a plasmid capable of expressing the fish granzyme B gene;

[0011] Use the plasmid to infect cells, and isolate and purify the recombinant protein of fish granzyme B from the infected cells;

[0012] Use the recombinant protein of fish granzyme B as an immunogen for animal immunization;

[0013] After animal immunization, cell fusion, cell screening and cloning are carried out to obtain the hybridoma cell line 2C8H3.

[0014] In a preferred embodiment of the present invention, the cells for infection include 293T cells.

[0015] In a preferred embodiment of the present invention, the process of isolating and purifying the recombinant protein of fish granzyme B includes:

[0016] Collect the culture supernatant from the infected cells, centrifuge to obtain the supernatant containing the eukaryotic recombinant protein of Granzyme B;

[0017] Add Protein G Agarose to the supernatant, incubate, centrifuge, wash Protein G to remove the unbound proteins;

[0018] Elute the eukaryotic recombinant protein of Granzyme B with acidic Glycine-HCl solution and neutralize it with alkaline Tris-HCL solution;

[0019] Put the eluted target protein into pre-cooled PBS for full dialysis, collect the supernatant, centrifuge to an appropriate volume to obtain the purified recombinant protein of fish Granzyme B.

[0020] In a third aspect, the present invention discloses a monoclonal antibody against fish Granzyme B secreted by the hybridoma cell line 2C8H3 as described in the first aspect.

[0021] In a fourth aspect, the present invention discloses a method for preparing the monoclonal antibody as described in the third aspect, comprising the following steps:

[0022] Use the purified recombinant protein of fish Granzyme B as an antigen to immunize mice until the mice produce antibodies;

[0023] Fuse the spleen leukocytes of the immunized mice with SP2 cells, screen out the hybridoma cells that can stably secrete antibodies, and perform subcloning on the positive cell wells;

[0024] Screen out the antibodies from the antibodies produced by the hybridoma cells that can specifically bind to the Granzyme B protein secreted by fish T lymphocytes, which are the monoclonal antibodies.

[0025] In a fifth aspect, the present invention discloses the application of the monoclonal antibody as described in the third aspect in the study of fish adaptive immune response.

[0026] In a sixth aspect, the present invention discloses the application of the monoclonal antibody as described in the third aspect in the preparation of a kit for detecting Streptococcus agalactiae.

[0027] In a seventh aspect, the present invention discloses the application of the monoclonal antibody as described in the third aspect in specifically recognizing fish Granzyme B protein.

[0028] In an eighth aspect, the present invention discloses the application of the monoclonal antibody as described in the third aspect in the preparation of fish disease prevention and treatment reagents / drugs.

[0029] Furthermore, the fish disease prevention and treatment reagent / drug includes a fish disease prevention and treatment vaccine.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention purifies the tilapia granzyme B protein through a eukaryotic expression system, immunizes mice with this as an antigen, and on this basis constructs a screening and identification method combining flow cytometry, semi-quantitative detection and enzyme-linked immunosorbent assay, and successfully prepares a monoclonal antibody that can specifically recognize the anti-Nile tilapia granzyme B molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram for purifying granzyme B protein;

[0033] Figure 2 Flow cytometry analysis results of specific cell populations after 2C8 fusion cell supernatant-bound lymphocytes were stimulated with P+I for 4 hours;

[0034] Figure 3 ELISA analysis results of 2C8 fusion cell culture supernatant binding to granzyme B protein;

[0035] Figure 4 Flow cytometry analysis results of specific cell populations after 2C8H3 fusion cell supernatant-bound lymphocytes were stimulated with P+I for 4 hours after subcloning;

[0036] Figure 5 Electrophoresis patterns of each gene in granzyme B positive and negative cells;

[0037] Figure 6 Detection of the change in the proportion of granzyme B positive cell populations in anti-granzyme B monoclonal antibody-stimulated or non-stimulated anti-tilapia CD3 / CD28 monoclonal antibody and in the Streptococcus agalactiae-infected group and the control group;

[0038] Figure 7 Expression of granzyme B at the protein level after anti-granzyme B monoclonal antibody specifically binds to the protein sample and is stimulated with PHA for 12 hours;

[0039] Figure 8 Detection of the proportion of granzyme B positive cell populations in lymphocytes of tissues such as head kidney, peripheral blood and spleen by anti-granzyme B monoclonal antibody. DETAILED DESCRIPTION OF THE INVENTION

[0040] The following will combine the drawings and examples to give a rigorous and detailed description of the present invention. It is emphasized that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] In the following content, the fish include teleosts, preferably tilapia, and particularly preferably Nile tilapia (Oreochromis niloticus).

[0042] Example 1

[0043] This example provides a hybridoma cell line 2C8H3 that secretes anti-fish granzyme B monoclonal antibody. The hybridoma cell line 2C8H3 was deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with the deposit number CCTCC NO: C2024141. Among them, the fish is Nile tilapia (Oreochromis niloticus).

[0044] The preparation method of the above hybridoma cell line 2C8H3 includes the following steps:

[0045] S1: Construct a plasmid capable of expressing the fish granzyme B gene;

[0046] The main method is as follows:

[0047] Synthesis of Nile tilapia granzyme B gene:

[0048] ① Obtain the Nile tilapia granzyme B gene fragment sequence (accession number: XM_003439678.4) from NCBI. Synthesize the gene fragment by Sangon Biotech Co., Ltd., and ligate the gene fragment into the pINFUSE-mIgG2b-Fc2 vector using BamHⅠ and XhoⅠ as restriction enzyme sites to obtain a complete plasmid (Granzyme B-pINFUSE-mIgG2b-Fc2) for subsequent experiments.

[0049] S2: Infect cells with the plasmid and isolate and purify the recombinant protein of fish granzyme B from the infected cells;

[0050] The main method is as follows:

[0051] Eukaryotic expression and purification of Granzyme B protein:

[0052] ② Uniformly seed 6.6×10 6 293T cells in a 10 cm cell culture dish and culture overnight. When the cell confluence reaches 80%-90%, replace the medium with 9 mL of DMEM containing 25 mM chloroquine (1% double antibody, 10% FBS);

[0053] ② Transfection: Add 30 μg of Granzyme B-pINFUSE-mIgG2b-Fc2 plasmid and 150 μL of 2.5 M CaCl2 into 1500 μL of ddH2O, gently pipette to mix, and slowly add 1500 μL of 2×HEPES-Buffer while pipetting. Prepare the transfection system, mix well, carefully add it dropwise to the above-mentioned 293T cells, and continue to culture in a 37°C constant temperature incubator;

[0054] ③ Protein purification: After 48 h of transfection, collect the culture supernatant of 293T cells, centrifuge at 1200 rpm for 10 min to obtain the supernatant containing the eukaryotic recombinant protein of Granzyme B; Add 1 mL of Protein G Agarose to the above supernatant, incubate overnight on a shaker at 4°C; Centrifuge at 1500 rpm for 1 min, wash Protein G with 10 mL of PBS, and repeat 6 times to thoroughly wash away unbound proteins; Elute the eukaryotic recombinant protein of Granzyme B with 10 mL of 0.1 M Glycine-HCl (pH 2.8), and add 1 mL of 1 mM Tris-HCL (pH 8.5) for neutralization; Place the eluted target protein in pre-cooled PBS at 4°C and dialyze thoroughly for 24 h; Collect the supernatant, transfer it into a 10 KDa ultrafiltration tube, centrifuge at 4000 rpm at 4°C to an appropriate volume, and detect the protein concentration using a BCA protein quantification detection kit. Aliquot the detected protein and store it frozen at -80°C in a refrigerator.

[0055] ④ Dilute the obtained protein, add 5×SDS loading buffer, heat in a boiling water bath for 5 min, and detect the protein purity using 12% SDS-PAGE;

[0056] As Figure 1 shown, the recombinant protein of tilapia granzyme B was purified, and its size was between 35 - 40 KDa.

[0057] S3: Use the recombinant protein of fish granzyme B as an immunogen for animal immunization;

[0058] The main method is as follows:

[0059] Animal immunization:

[0060] Use the purified protein as an antigen to immunize 6-week-old BALB / c mice, for a total of 5 immunizations. The second immunization is carried out 2 weeks after the primary immunization, and then immunizations are carried out once every week. For the primary and secondary immunizations, emulsify 100 μg of purified protein with complete Freund's adjuvant and incomplete Freund's adjuvant respectively, and inject the mice intraperitoneally. For the last three immunizations, inject 100 μg of protein into the mice via the tail vein.

[0061] S4: After immunizing the animals, cell fusion, cell screening, and cloning are carried out to obtain the hybridoma cell line 2C8H3.

[0062] The following method is mainly adopted:

[0063] Cell fusion:

[0064] ① One week before fusion, resuscitate and passage-culture SP2 cells, check the cell growth condition daily and passage as needed. Transfer them to a T75 cell culture flask 2 days before fusion and change the culture medium 12 h before fusion;

[0065] ② On the 5th day after the last immunization, carry out cell fusion. First, take out the thymus of a non-immunized 4-week-old healthy mouse, wash it with RPMI-1640 medium, then grind it on a 200-mesh sterile sieve, collect the cells with RPMI-1640 and place them in a large centrifuge tube, centrifuge, and resuspend the thymus cells with RPMI-1640 containing HAT; then sacrifice the immunized mouse, take out its spleen, grind, filter, and resuspend the spleen cells with RPMI-1640 as described above, discard the large precipitates, obtain the corresponding cell suspension, and temporarily store the thymus cells in a 37 °C incubator;

[0066] ③ Mix the above spleen cell suspension with SP2 cells, then place them in a large centrifuge tube and centrifuge at 1200 rpm for 8 min, discard the supernatant, gently tap the bottom of the large centrifuge tube with the wrist to loosen the cell precipitate at the bottom, and then place the large centrifuge tube in a 37 °C water bath;

[0067] ④ Slowly add 1 mL of PEG (1.8 g PEG, 2 mL RPMI-1640 medium, 200 μL DMSO) dropwise to the loosened mixed cell precipitate within 90 s, let it stand for 90 s, and then continue to slowly add 10 mL of RPMI-1640 dropwise within 5 min. After the addition is completed, centrifuge at 800 rpm for 6 min and discard the supernatant;

[0068] ⑤ Add 3 mL of RPMI-1640 to resuspend the cell precipitate, and add it to the thymus cell suspension in ② above. Drop the mixed cell suspension evenly into a 96-well cell culture plate, about 100 μL per well;

[0069] ⑥ Place the above cell culture plate in a 37 °C incubator. After about 10 - 14 days, aspirate the supernatant of the fused cells and add 200 μL of HAT-RPMI-1640 medium containing 10% FBS to continue the culture;

[0070] ⑦ Screening can be carried out again after about 2 - 3 days, and the obtained supernatant is used for subsequent experiments;

[0071] Screening of monoclonal antibody-positive fused cells:

[0072] ① Place the spleen of Nile tilapia in L15 medium for washing. After grinding and filtering, add it to a percoll separation solution with densities of 52% and 34% (4 mL of 34% solution in the upper layer and 4 mL of 52% solution in the lower layer). Set the acceleration and deceleration to 0 and centrifuge at 500 g for 30 min.

[0073] ② After centrifugation, a circular white blood cell layer appears at the stratification. Slowly aspirate the white blood cells suspended in the separation layer, wash them with L15 medium and centrifuge for 3 min. Discard the supernatant, then resuspend with DMEM medium (DMEM medium + 10% FBS + 1% double antibody), place it in an incubator at 28 °C for 30 min of static culture, and then add 1 μL of P+I (PMA + ionomycin, V:V = 1:1) to stimulate the cells. After 4 h, blow up the cells, centrifuge and wash, then resuspend with PBS and add them to a 96-well V-bottom plate.

[0074] ③ After centrifugation at 2500 rpm for 3 min, discard the supernatant, fix with BD fixative on ice for 30 min, then resuspend the cells with 100 μL of fused cell supernatant. For negative wells, add 100 μL of SP2 cell supernatant and incubate on ice for 30 min at the same time. After centrifuging the cells, wash them twice with 200 μL of washing Buffer.

[0075] ④ Resuspend the centrifuged cells with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold, incubate on ice for 30 min in the dark, and after centrifuging the cells, wash them twice with 200 μL of washing Buffer.

[0076] ⑤ After washing, resuspend the cells with 200 μL of washing Buffer, filter, and then detect and analyze by flow cytometry.

[0077] As Figure 2 shown, it was found by flow cytometry detection that the antibody secreted by the hybridoma cells (fused cells of spleen white blood cells and SP2) in well 2C8 could bind to the protein in a certain cell population in Nile tilapia white blood cells, and the proportion of this population in Nile tilapia spleen lymphocytes after 4 h of P+I stimulation was approximately 7.22%.

[0078] Determine the binding of the antibody secreted by 2C8 cells to tilapia granzyme B protein by ELISA method:

[0079] ① Dissolve 5 μg of recombinant protein granzyme B in 50 mM sodium carbonate-sodium bicarbonate buffer (pH = 9.8), add 100 μL of the coating solution containing recombinant protein granzyme B to each well to coat the ELISA plate, and incubate overnight at 4 °C;

[0080] ② After the incubation, wash the ELISA plate 3 times with PBST (PBS + 0.5‰ tween 20), 5 min each time. Then add 200 μL of 1% BSA to each well and incubate for 1 h in a 37 °C incubator;

[0081] ③ After the incubation, wash the ELISA plate 3 times with PBST, 5 min each time. Then add 100 μL of the primary antibody (i.e., the supernatant of the fused cells) to each well and incubate for 1 h in a 37 °C incubator;

[0082] ④ After the incubation, wash the ELISA plate 3 times with PBST, 5 min each time. Then add 100 μL of alkaline phosphatase-labeled (AP) goat anti-mouse IgG (1:3000) to each well and incubate for 1 h in a 37 °C incubator;

[0083] ⑤ After the incubation, wash the ELISA plate 3 times with PBST, 5 min each time. Then add 100 μL of 50 mM sodium carbonate-sodium bicarbonate buffer (pH = 9.8) containing 0.1% (w / v) p-nitrophenyl phosphate (pNPP) and 0.5 mM MgCl 2 to each well, place it in the dark for 30 min for color development. After the color development is completed, add 50 μL of 2 M NaOH to each well to stop the color development, and measure the absorbance at OD 405 .

[0084] As Figure 3 shown, the expression level of granzyme B gene in the positive well 2C8 is relatively high, while the negative well basically does not express granzyme B, indicating that this monoclonal antibody can specifically bind to the granzyme B protein secreted by Nile tilapia T lymphocytes.

[0085] Subclone positive hybridoma cells:

[0086] ① Take the thymus of a healthy 4-week-old mouse without treatment. In the same way as the above treatment, first wash it with RPMI-1640 medium, grind it aseptically, centrifuge it, and then resuspend it with the medium to obtain a thymocyte suspension. Centrifuge it at 1000 rpm for 5 min, and resuspend the precipitate with 10 mL of RPMI-1640 and mix well;

[0087] ② Suspend the hybridoma cells (the fused cells of spleen leukocytes and SP2) in the 2C8 well, perform gradient dilution with RPMI-1640 medium, and count the diluted cells with a hemocytometer;

[0088] ③ After counting, add about 100 hybridoma cells to the above-mentioned thymocyte suspension, mix evenly, and slowly drip it into a 96-well cell culture plate, with a volume of about 100 μL per well;

[0089] ④ Place the culture plate in an incubator at 37 °C. After about 10 - 14 days, aspirate the hybridoma supernatant and detect it by flow cytometry according to the above method;

[0090] As Figure 4 shown, after cloning the cells in the 2C8 well, the obtained hybridoma cell line can stably secrete antibodies, and this antibody can still bind to the proteins in a certain cell population in Nile tilapia leukocytes, and the ratio is stable. This hybridoma cell line is named 2C8H3;

[0091] Identify the antibody secreted by 2C8H3 cells as a monoclonal antibody against tilapia granzyme B by semi - quantitative method:

[0092] ① Take the spleen of Nile tilapia according to the above method. After washing, grinding, and filtering, add it to the percoll separation solution. After centrifugation, aspirate the leukocyte layer suspended in the separation layer. After washing and centrifuging with L15 medium, resuspend it with L15 medium, and then transfer it to a 2 mL EP tube;

[0093] ② After centrifuging at 2500 rpm for 3 min, discard the supernatant, fix it on ice with BD fixative for 30 min, then wash it twice with washing buffer, suspend the leukocyte precipitate with 1.5 mL of 2C8H3 culture supernatant, incubate it on ice for 30 min, and wash it twice with 1 mL of washing buffer after centrifugation;

[0094] ③ Suspend the centrifuged cells with 1.5 mL of anti - mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000 - fold, incubate it statically on ice for 30 min in the dark, and wash the cells twice with 1 mL of washing buffer after centrifugation;

[0095] ④ Suspend the cells with 1 mL of washing buffer and then sort them. Collect the positive and negative cell populations respectively. At least 2×10 6 cells should be collected for the positive cells;

[0096] ⑤ Use the Trizol method to extract the total RNA of positive and negative cells, reverse - transcribe to obtain cDNA templates, and amplify the gene fragments of Nile tilapia β - actin, granzyme B, CD3, and IgM through PCR reaction. The primer sequences are shown in the following table;

[0097] Primer information

[0098]

[0099] ⑥Mix the PCR product with 6×loading buffer, and then add it to a 2% agarose gel for electrophoresis. Adjust the loading amount of the PCR product to change the band brightness. After making the amount of β-actin in the positive and negative cell populations the same, re-electrophorese and then detect the expression levels of each gene.

[0100] As Figure 5 shown, the expression level of the granzyme B gene in the positive cell population is extremely high, while the negative population does not express granzyme B, and most of the positive population expresses CD3ε but does not express IgM, indicating that granzyme B + The positive cell population is mostly T lymphocyte population, proving that this monoclonal antibody can specifically bind to the granzyme B protein secreted by Nile tilapia T lymphocytes.

[0101] In this example, the granzyme B protein was purified and the obtained protein was used as an antigen to immunize mice. After the mice produced antibodies, the spleen cells of the immunized mice were fused with SP2 cells, and hybridoma cells that could stably secrete antibodies were screened by flow cytometry, and the cell wells showing positive by flow cytometry were subcloned. By ELISA and semi-quantitative screening methods, it was determined that the antibody produced by the 2C8H3 strain cells could specifically bind to the granzyme B protein secreted by Nile tilapia T lymphocytes and could specifically recognize Nile tilapia granzyme B + cells.

[0102] Example 2

[0103] This example provides an application of the anti-fish granzyme B monoclonal antibody secreted by the hybridoma cell line 2C8H3 in Example 1 in the study of fish adaptive immune response.

[0104] Use the anti-Nile tilapia granzyme B monoclonal antibody to detect the number of granzyme B + lymphocytes in T cells of Nile tilapia spleen lymphocytes after stimulation with anti-Nile tilapia CD3 / 28 monoclonal antibody, which includes the following steps:

[0105] The applicant has previously prepared monoclonal antibodies against tilapia CD3 and CD28. For example, the preparation of a monoclonal antibody against tilapia CD3ε was disclosed in a Chinese patent with the publication number CN112501130A, and the preparation of a monoclonal antibody against tilapia CD28 was disclosed in a Chinese patent with the publication number CN116355861A. In vitro, lymphocytes were stimulated with antibodies to simulate the two signals of T cell activation. A healthy tilapia was sacrificed, and its spleen was removed, ground and filtered, and spleen lymphocytes were obtained by the above density gradient centrifugation method. The lymphocytes were resuspended in 1 mL of DMEM medium. 500 μL was taken as a control, and 2 μg of CD3 / 28 monoclonal antibody was added to another 500 μL of lymphocytes. At the same time, 1 μL of Golgi plug was added to both wells, and the medium was supplemented to 1 mL, and the cells were stimulated and cultured in an incubator at 28 °C. After 12 h of stimulation, the lymphocytes were taken into a 96-well V-bottom plate and centrifuged at 2500 rpm for 3 min. After the CD3 antibody was diluted at 1:400, it was added to the wells, and the cells were resuspended with 100 μL per well and stained for 30 min. After centrifugation and washing, the centrifuged cells were resuspended with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold, and incubated on ice for 30 min in the dark. After the staining was completed, the cells were centrifuged, fixed with BD fixative on ice for 30 min, centrifuged again, washed twice with washing buffer, and then the cells were resuspended with 100 μL of the anti-tilapia granzyme B-FITC monoclonal antibody labeled in Example 2 and incubated on ice for 30 min. After centrifugation, the cells were washed twice with 200 μL of washing buffer; after centrifugation, the cells were washed twice with 200 μL of washing buffer; after the cells were resuspended with 200 μL of washing buffer, they were filtered through a 200-mesh sieve and subjected to flow cytometry detection.

[0106] As Figure 6 shown above, by flow cytometry analysis, after 12 h of stimulation of spleen lymphocytes with CD3 / 28 monoclonal antibody, the proportion of granzyme B + cells in T lymphocytes increased from 7.46% before stimulation to 14.7% after stimulation, indicating that the proportion of T lymphocytes expressing granzyme B increased sharply after stimulation, and granzyme B responded to the activation of T cells.

[0107] Example 3

[0108] This example provides an application of the fish granzyme B monoclonal antibody secreted by the hybridoma cell line 2C8H3 in Example 1 in detecting Streptococcus agalactiae infection.

[0109] Detection of Nile tilapia granzyme B after Streptococcus agalactiae infection using anti-Nile tilapia granzyme B monoclonal antibody + To detect the number of lymphocytes, the following steps are included:

[0110] Add the bacterial solution to 5 mL of BHI medium at a ratio of 1:1000. After the medium becomes turbid, adjust the concentration of the bacterial solution using an enzyme-linked immunosorbent assay (ELISA) plate. When the concentration of the bacterial solution is adjusted to OD 600 = 1, dilute the bacterial solution 40-fold. Inject 200 μL of the diluted Streptococcus agalactiae into the abdominal cavity of each healthy Nile tilapia. The control group is injected with the same dose of PBS. On the 5th day after infection, remove the spleen tissues of Nile tilapia in different treatment groups. One day before euthanasia, inject 100 μL of BFA into the Nile tilapia in the two treatment groups. Obtain white blood cells using the above-mentioned percoll centrifugation method and resuspend them in FACS Buffer. Add the cell suspension to a 96-well V-bottom plate, centrifuge at 2500 rpm for 3 min, and discard the supernatant. Dilute the CD3 antibody 1:400 and add 100 μL to each well. Resuspend the cells and stain for 30 min. Then, resuspend the centrifuged cells with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold, and incubate on ice in the dark for 30 min. After staining, centrifuge, fix with BD fixing solution on ice for 30 min, wash twice with FASC buffer, centrifuge again, wash twice with washing buffer, resuspend the cells with 100 μL of the anti-Nile tilapia granzyme B-FITC monoclonal antibody labeled in Example 2, incubate on ice for 30 min, centrifuge the cells, and wash twice with 200 μL of washing Buffer; centrifuge the cells and wash twice with 200 μL of washing Buffer; resuspend the cells with 200 μL of washing Buffer, filter through a 200-mesh sieve, and perform flow cytometry analysis.

[0111] As Figure 6 shown below, by flow cytometry analysis, 5 days after Streptococcus agalactiae infection, the proportion of granzyme B + cells in T lymphocytes increased from 8.6% before infection to 42.5% after infection, indicating that the proportion of lymphocytes expressing granzyme B increased sharply after infection and participated in the antibacterial immunity of Nile tilapia.

[0112] Example 4

[0113] This example provides an application of the anti-fish granzyme B monoclonal antibody secreted by the hybridoma cell line 2C8H3 in Example 1 in specifically recognizing fish Granzyme B protein.

[0114] Detection of the specific recognition of Nile tilapia granzyme B protein by anti-Nile tilapia granzyme B monoclonal antibody. The experimental steps are as follows:

[0115] Isolate white blood cells from the spleen of healthy Nile tilapia, resuspend them with 1 mL of DMEM medium, and divide them equally into two wells. Add 500 μL of medium to one well for culture, and add 500 μL of medium and 2 μL of PHA to the other well for stimulation for 12 h. After the stimulation is completed, blow and suspend the cells, centrifuge at 8000 rpm for 3 min, discard the supernatant, add 80 μL of NP-40 lysis buffer (1% protease inhibitor, 1‰ PMSF, 1% phosphatase inhibitor) to each tube to resuspend the cells, and lyse the cells on ice for 30 min; centrifuge at 12000 rpm at 4 °C for 10 min, take the supernatant, add 20 μL of 5×SDS loading buffer, mix well, and boil the sample in a boiling water bath for 10 min to obtain two groups of cell samples;

[0116] Mix 20 μL of granzyme B protein with 5 μL of 5×SDS loading buffer, and boil the sample in a boiling water bath for 10 min to obtain a protein sample;

[0117] Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the protein sample and the cell sample, and add 10 μL of the sample to each well; first maintain a constant current of 40 mA. When the sample runs to the boundary between the stacking gel and the separating gel, adjust the current to 80 mA, and run the gel to the appropriate position according to the position of the maker and then stop;

[0118] Open the gel plate, remove the stacking gel after taking out the gel, then attach the gel to the NC membrane with a transfer device, and place it in the transfer solution (transfer solution: 10% transfer buffer, 70% ultrapure water, 20% methanol), and run at a constant voltage of 100 V for 2 h. During the transfer operation, the transfer device is placed in an ice-water bath throughout; after all the samples are transferred to the NC membrane, place the NC membrane in 15 mL of PBST solution containing 4% skim milk powder and block it at room temperature for 1 h; then wash it three times with PBST, 10 min each time. Finally, cut the membrane according to the size of the target protein and put it into the antibody supernatant secreted by the granzyme B fusion cells respectively, and incubate overnight at 4 °C;

[0119] After incubation, wash the membrane 3 times with PBST, 10 min each time;

[0120] After washing, add anti-mouse Alexa Fluor 680 to 10 mL of PBST containing 4% skim milk powder at a ratio of 1:10000, and incubate at room temperature for 1 h; then wash it 3 times with PBST, 10 min each time; scan the NC membrane using an Odyssey CLX imaging system.

[0121] As Figure 7 shown, the anti-Nile tilapia granzyme B antibody can specifically bind to the Nile tilapia granzyme B protein, and after 12 h of PHA stimulation, the protein expression level of granzyme B is up-regulated. This indicates that the obtained granzyme B antibody has the ability to specifically bind to the protein, and the expression of granzyme B increases after T cell activation.

[0122] Example 5

[0123] Application of monoclonal antibody granzyme B in detecting the tissue distribution of granzyme B + lymphocyte populations in fish.

[0124] granzyme B + To detect the distribution of lymphocytes in different tissues of Nile tilapia, the experimental steps are as follows:

[0125] Take a healthy Nile tilapia, collect peripheral blood, liver, gill, head kidney and spleen respectively, and isolate white blood cells according to the method in Example 1.

[0126] Among them, when isolating peripheral blood white blood cells, first use a 5 mL syringe to take 2 mL of anticoagulant (15 mM sodium citrate, 450 mM NaCl, 0.1 M glucose, 10 mM EDTA, pH 7.0), expel air bubbles, then use the syringe to draw peripheral blood from the caudal vein and mix well. Centrifuge at 2500 rpm for 3 min, discard the supernatant, resuspend the cell pellet with L15 medium, and isolate white blood cells using the above-mentioned percoll density gradient separation method;

[0127] Resuspend the white blood cells of each tissue with 1 mL of FACS buffer respectively. Add 100 μL of cell suspension to each well of a 96-well V-bottom plate, centrifuge at 2500 rpm for 3 min, and discard the supernatant; Fix with BD fixative on ice for 30 min, centrifuge, wash twice with washing buffer, then resuspend the cells with 100 μL of the supernatant of the anti-tilapia granzyme B fusion cells in Example 2. Add 100 μL of SP2 cell supernatant to the wells of the negative control group, and incubate on ice for 30 min. Centrifuge the cells and wash twice with 200 μL of washing buffer; Resuspend the centrifuged cells with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold, and incubate on ice for 30 min in the dark. Centrifuge the cells and wash twice with 200 μL of washing buffer; Resuspend the cells with 200 μL of washing buffer and filter, and perform flow cytometry.

[0128] As Figure 8 shown, the percentages of granzyme B lymphocytes in peripheral blood, liver, gill, head kidney and spleen lymphocytes were 21.2%, 14.1%, 23.0%, 30.7% and 8.34% respectively, indicating that the anti- Tilapia granzyme B monoclonal antibody could identify granzyme B + cell populations in multiple tissues. + Cell population.

[0129] In summary, the present invention has established a complete eukaryotic expression system and purification system. Based on this system, the project prepared and purified the eukaryotic recombinant protein of fish Granzyme B to immunize mice, and constructed a screening and identification mechanism combining flow cytometry, semi-quantitative detection and ELISA detection, and successfully prepared the monoclonal antibody against fish granzyme B, providing important tools and technical support for the research on fish adaptive immunity, especially the research on fish cytotoxic T cells, and also providing technical support and theoretical basis for the prevention and control of fish diseases and the evaluation of vaccine effects.

[0130] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hybridoma cell line 2C8H3 secreting anti-fish granzyme B monoclonal antibody, characterized in that: The hybridoma cell line 2C8H3 is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2024141 and a deposit date of May 9, 2024.

2. A method for preparing the hybridoma cell line 2C8H3 according to claim 1, characterized in that: The steps include: Construct a plasmid that can express fish granzyme B gene; Infecting cells with the plasmid, and isolating and purifying the recombinant protein of fish granzyme B from the infected cells; Animal immunization was performed using the recombinant protein of fish granzyme B as an immunogen; After animal immunization, cell fusion, cell screening and cloning are performed to obtain the hybridoma cell line 2C8H3.

3. The preparation method according to claim 2, characterized in that: The process of isolating and purifying the recombinant protein of fish granzyme B includes: The culture supernatant of the infected cells was collected and centrifuged to obtain the supernatant containing Granzyme B eukaryotic recombinant protein; Add Protein G agarose to the supernatant, incubate, centrifuge, wash Protein G, and wash away unbound proteins; Granzyme B eukaryotic recombinant protein was eluted with acidic Glycine-HCl solution and neutralized with alkaline Tris-HCl solution; The eluted target protein was placed in pre-cooled PBS for full dialyzation, the supernatant was collected, and centrifuged to an appropriate volume to obtain the purified recombinant protein of fish granzyme B.

4. An anti-fish granzyme B monoclonal antibody secreted by the hybridoma cell line 2C8H3 as claimed in claim 1.

5. A method for preparing a monoclonal antibody according to claim 4, characterized in that: The steps include: The purified recombinant protein of fish granzyme B was used as an antigen to immunize mice until the mice produced antibodies; The spleen leukocytes of immunized mice were fused with SP2 cells to screen hybridoma cells that could stably secrete antibodies, and the positive cell wells were subcloned; The antibodies produced by hybridoma cells that can specifically bind to the granzyme B protein secreted by fish T lymphocytes are screened out as monoclonal antibodies.

6. Use of the monoclonal antibody as claimed in claim 4 in the study of adaptive immune response in fish.

7. Use of the monoclonal antibody as claimed in claim 4 in preparing a kit for detecting Streptococcus agalactiae.

8. Use of the monoclonal antibody according to claim 4 in specifically identifying fish Granzyme B protein.

9. Use of the monoclonal antibody as claimed in claim 4 in the preparation of fish disease prevention and treatment reagents / medicines.

10. The use according to claim 9, characterized in that The fish disease prevention and treatment reagent / drug includes a fish disease prevention and treatment vaccine.

Images