Hybridoma cell strain secreting monoclonal antibody resisting tilapia CD122 protein and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain secreting a monoclonal antibody against a tilapia mossambica CD122 protein and application of the hybridoma cell strain 2D8A7. The hybridoma cell strain 2D8A7 is preserved in China Center for Type Culture Collection (CCTCC) on May 9, 2024, the preservation number is CCTCC NO: C2024142, and the preservation address is Wuhan University, Wuhan, China. According to the application, the active tilapia CD122 protein can be expressed on the cell surface, and the monoclonal antibody for resisting the tilapia CD122 is successfully prepared, so that a reference basis is provided for fish adaptive immunity research, fish disease prevention and control and vaccine effect evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of fish immunology, and specifically relates to a hybridoma cell line secreting monoclonal antibodies against tilapia CD122 protein, monoclonal antibodies, and their preparation methods and applications. Background Art

[0002] During the activation and proliferation of T cells, CD122 is the β subunit of the interleukin-2 / interleukin-15 (IL-2 / IL-15) receptor, mainly present on the surface of T cells and NK cells, and can be induced to express by cytokines such as IL-2. After IL-2 or IL-15 binds to its receptor complex, downstream signal transduction is mainly mediated by the intracellular segment of CD122. After tyrosine phosphorylation of the intracellular segment of CD122, it can serve as a docking point for STAT5, thereby activating the JAK-STAT signal transduction and participating in various biological function processes such as T cell activation, proliferation, and toxicity exertion. Therefore, CD122 is often considered a marker receptor on the surface of T cell activation and CD8+ T cells.

[0003] Due to the complexity of the transmembrane protein structure, it is still difficult to express a large amount of CD122 protein with a complete natural structure and original function in vitro at present, making it difficult to apply traditional monoclonal antibody preparation methods to the preparation process of monoclonal antibodies against CD122 protein on the cell surface. Further, due to the difficulty in preparing specific monoclonal antibodies against tilapia CD122 protein, problems such as the process and mechanism of T lymphocyte activation and toxicity function exertion in the adaptive immunity of tilapia cannot be studied in depth. Summary of the Invention

[0004] The present invention aims at the defects in the above-mentioned prior art and provides a hybridoma cell line secreting monoclonal antibodies against tilapia CD122 protein and its application. The present application can express active tilapia CD122 protein on the cell surface and successfully prepare monoclonal antibodies against tilapia CD122, providing a reference basis for fish adaptive immunity research, fish disease prevention and control, and vaccine effect evaluation.

[0005] The technical solutions provided by the present invention to solve the above technical problems are as follows:

[0006] On the one hand, a hybridoma cell line 2D8A7 (Hybridoma cell line 2D8A7) secreting monoclonal antibodies against tilapia CD122 protein is provided. The hybridoma cell line 2D8A7 was deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with the deposit number CCTCC NO: C2024142 and the deposit address being Wuhan University, Wuhan, China.

[0007] On the other hand, a method for preparing the above-mentioned hybridoma cell line 2D8A7 is also provided, which comprises the following steps:

[0008] Constructing a gene expression vector of tilapia T lymphocyte surface membrane protein CD122;

[0009] Preparing retrovirus;

[0010] Infecting NIH / 3T3 cells with the retrovirus;

[0011] Using the NIH / 3T3 cells infected with the retrovirus as an antigen for animal immunization;

[0012] Performing cell fusion using the immunized animal to obtain positive hybridoma cells;

[0013] And cloning the positive hybridoma cells.

[0014] On the other hand, a monoclonal antibody against tilapia CD122 protein secreted by the above-mentioned hybridoma cell line 2D8A7 is also provided.

[0015] On the other hand, a method for preparing the above-mentioned monoclonal antibody is also provided, which comprises the following steps:

[0016] Ascites preparation: Taking a mouse and intraperitoneally injecting sterile paraffin oil;

[0017] Resuspending the above-mentioned hybridoma cell line 2D8A7 and then intraperitoneally injecting the mouse;

[0018] Executing the mouse, collecting the ascites and centrifuging, and storing in aliquots;

[0019] Purifying the ascites to obtain the monoclonal antibody against tilapia CD122 protein.

[0020] On the other hand, an application of the above-mentioned monoclonal antibody in response to infection with Edwardsiella piscicida is also provided.

[0021] On the other hand, an application of the above-mentioned monoclonal antibody in detecting the distribution of CD122 protein in tilapia immune-related tissues is also provided.

[0022] Preferably, the immune-related tissues include one or more of peripheral blood, head kidney, liver and spleen.

[0023] On the other hand, an application of the above-mentioned monoclonal antibody in specifically recognizing tilapia CD122 protein is also provided.

[0024] On the other hand, an application of the above-mentioned monoclonal antibody in increasing the expression of tilapia CD122 protein is also provided.

[0025] On the other hand, there is also provided an application of the above monoclonal antibody in the preparation of fish disease prevention and treatment reagents / drugs.

[0026] Preferably, the fish disease prevention and treatment reagents / drugs include fish disease prevention and treatment vaccines.

[0027] In the present invention, the lentivirus infection method is adopted to transfer the plasmid containing the gene fragment of Nile tilapia CD122 into the mouse homologous NIH / 3T3 cell line, so that the active tilapia CD122 protein can be expressed on the cell surface, and a screening and identification mechanism combining flow cytometry, semi-quantitative detection and immunofluorescence technology is constructed. A monoclonal antibody against tilapia CD122 was successfully prepared, providing strong evidence for understanding CD122 as a marker molecule for early vertebrate T cell activation, and also providing a reference basis for fish adaptive immunity research, fish disease prevention and control, and vaccine effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a staining image of 293T cells;

[0029] Figure 2 It is a staining image of NIH / 3T3 cells;

[0030] Figure 3 It is the flow cytometry analysis result of NIH / 3T3 cells after being infected with retrovirus;

[0031] Figure 4 It is the flow cytometry analysis result of hybridoma cell line 2D8A7 binding to specific cell populations in white blood cells, where (a) is a histogram and (b) is a density plot;

[0032] Figure 5 It is the electrophoresis pattern of each gene in CD122 positive and negative cell populations;

[0033] Figure 6 It is the staining image of the antibody secreted by hybridoma cell line 2D8A7 binding to CD122 protein;

[0034] Figure 7 It is the flow cytometry analysis result of anti-tilapia CD122 monoclonal antibody detecting the infected group and the control group of Edwardsiella piscicida, where (a) is a histogram and (b) is a density plot;

[0035] Figure 8 It is the flow cytometry analysis result of anti-tilapia CD122 monoclonal antibody detecting lymphocytes in head kidney, peripheral blood, liver and spleen;

[0036] Figure 9 It is the electrophoresis pattern of Western blot detecting the binding of anti-tilapia CD122 monoclonal antibody to specific proteins in lymphocytes; Specific embodiments

[0037] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] Example 1:

[0039] This embodiment provides a hybridoma cell line 2D8A7 (Hybridoma cell line 2D8A7) that secretes antibodies against Oreochromis niloticus CD122 protein. The hybridoma cell line 2D8A7 was deposited with the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with the deposit number CCTCC NO: C2024142, and the deposit address is Wuhan University, Wuhan, China. The Oreochromis niloticus is Oreochromis niloticus.

[0040] Furthermore, the preparation method of the above-mentioned hybridoma cell line 2D8A7 includes the following steps:

[0041] Step 1: Construction of the expression vector of Oreochromis niloticus T lymphocyte surface membrane protein CD122 gene, which specifically includes the following steps:

[0042] 1. Obtain the Oreochromis niloticus CD122 gene fragment sequence (accession number: XM_019360337.2) from the NCBI database. The gene fragment was cloned by Sangon Biotech Co., Ltd. Using XhoⅠ and EcoRⅠ as restriction enzyme sites, the cloned CD122 gene fragment was ligated into the pMSCV vector.

[0043] 2. Introduce the pMSCV vector ligated with the CD122 gene fragment into the glycerol bacteria strain, and then expand the culture of the glycerol bacteria strain in 30 mL of LB liquid medium containing ampicillin resistance, and extract the retroviral expression vector containing the CD122 gene fragment from it, that is, the pMSCV-CD122 plasmid.

[0044] Step 2: Preparation of retrovirus, which specifically includes the following steps:

[0045] 1. Resuscitate 293T cells with DMEM complete medium (DMEM medium + 10% FBS + 1% double antibody, by weight), and culture them in a 6 cm diameter culture dish. After they grow to cover the culture dish, passage them.

[0046] At passage, aspirate the cell culture waste liquid in the culture dish. Wash the remaining cells with 3 mL of PBS. After discarding the PBS, add 1 mL of trypsin, shake well, incubate in a 37°C incubator for 30 s, discard the trypsin, then blow down and resuspend the cells with 3 mL of DMEM medium, and then transfer them to a 10-cm culture dish for culture;

[0047] After the cells are passaged three times, digest the 293T cells with trypsin, then centrifuge at 1000 rpm for 3 min, discard the supernatant. After blowing and resuspending the cells with DMEM medium, count them using a hemocytometer, and then inoculate 2.2×10 6 cells on a 6-cm diameter culture dish, and make up to 3 mL with DMEM medium for cell culture;

[0048] 2. Conduct transfection experiments 10 h after culturing, which specifically includes the following:

[0049] 2.1 Prepare the transfection system solution. The transfection system solution includes 10 μg of pMSCV-CD122 plasmid, 2 μg of pCMV-VSV-G, 5 μg of pCL-Eco plasmid, 50 μL of 2.5 M CaCl 2 solution, and finally add sterilized water to make up to 500 μL. Then gently blow the mixture with a dropper and slowly add 500 μL of 2×HEBS buffer;

[0050] 2.2 Discard the DMEM medium for cell culture after three passages, add 3 mL of DMEM complete medium containing 25 μM chloroquine, then dropwise add 1 mL of the above transfection system solution, and then place it in a 37°C incubator for 6 - 8 h for transfection. As Figure 1 shown, green fluorescence can be observed in the 293T cells after transfection under a fluorescence microscope;

[0051] 2.3 After culturing for 6 - 8 h, discard the culture medium supernatant, replace it with 3 mL of DMEM complete medium, and then replace the DMEM complete medium every 12 h. The volume of the DMEM complete medium replaced each time is 3 - 4 mL. After culturing the cells for 48 h, collect the supernatant, centrifuge the obtained supernatant at 1200 rpm for 8 min, collect the supernatant again, and conduct aliquoting, and freeze it in an -80°C refrigerator. The supernatant obtained by collecting again is the retrovirus suspension.

[0052] Step three: Infection of NIH / 3T3 cells, which specifically includes the following steps:

[0053] 1. Resuscitate NIH / 3T3 cells, then culture them in a 6-cm culture dish and passage them 3 times (the passage method can be the same as that described in Section 1 of Step 2). After passage, wash the cells with PBS, then add 1 mL of trypsin and digest at 37 °C for 30 s. Use DMEM medium to blow down all the adherent cells and resuspend them. Transfer the resuspended solution to a 15-mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, and retain the cell pellet. After resuspending the cell pellet with DMEM medium, blow the cells evenly, count them using a hemocytometer, and then inoculate 2×10 5 NIH / 3T3 cells in a 10-cm-diameter culture dish, and make up to 8 mL with DMEM medium for cell culture;

[0054] 2. After culturing for 12 h, discard the DMEM medium, slowly add 6 mL of DMEM complete medium containing 5 μM polybrene to the adherent cells, and then slowly drip 600 μL of the above retrovirus suspension into the NIH / 3T3 cells. Gently shake to mix evenly and culture in a 37 °C incubator;

[0055] 3. After culturing for 6 h, discard the supernatant, add 8 mL of pre-warmed DMEM complete medium, and then change the DMEM complete medium every 12 h for a total of 3 times. The volume of the DMEM complete medium changed each time is 8 - 10 mL to ensure that polybrene is washed away;

[0056] After culturing for 48 h, discard the supernatant and retain the cell pellet. Digest the cell pellet with trypsin for 1 minute, resuspend it with 3 mL of DMEM medium, add the obtained resuspended solution to a 15-mL centrifuge tube, and make up to 10 mL with PBS. Centrifuge at 1200 rpm for 3 min, discard the supernatant, retain the cell pellet, resuspend the cell pellet with 10 mL of PBS, and then centrifuge at 1200 rpm for 3 min. Discard the supernatant and retain the cell pellet. Then resuspend the cell pellet obtained from the last centrifugation in 200 μL of PBS.

[0057] As Figure 2 shown, it can be seen through fluorescence microscopy that the NIH / 3T3 cells carry green fluorescence, indicating that the retrovirus containing the Nile tilapia CD122 gene has successfully infected the NIH / 3T3 cells. And as Figure 3 shown, it can be known through flow cytometry analysis that the infection efficiency of NIH / 3T3 cells is relatively high, reaching 56.9%.

[0058] Step Four: Animal Immunization, which specifically includes the following steps:

[0059] Using NIH / 3T3 cells after retroviral infection as antigens, 6-week-old BALB / c mice were immunized by intraperitoneal injection, a total of 4 times. The second immunization was carried out 2 weeks after the primary immunization, and then immunization was carried out once every week. Each mouse was injected with about 1.5×10 7 NIH / 3T3 cells each time.

[0060] Step 5: Cell fusion, which specifically includes the following steps:

[0061] 1. Five days after the fourth immunization, take the thymus of 6-week-old healthy mice that have not been immunized, wash, grind, and centrifuge it with RPMI-1640 medium, then resuspend the thymus cells with HAT-RPMI-1640 RPMI-1640, discard large pieces of tissue, obtain a thymocyte suspension, and place it in a 37°C incubator for subsequent experiments. Also, take the spleen of the mice immunized as described above, wash, grind, and centrifuge it with RPMI-1640 RPMI-1640, discard large pieces of tissue, and then resuspend the spleen cells with RPMI-1640 medium to obtain a spleen cell suspension;

[0062] 2. Resuscitate P3-X63-Ag8U1 (abbreviated as P3U1) cells one week before fusion. Two days before fusion, transfer P3U1 cells with an appropriate density to a culture flask and change the culture medium 12 h before fusion;

[0063] 3. Fuse the above spleen cell suspension with the resuscitated and passaged P3U1 cells to obtain a P3U1-spleen cell suspension. Place the P3U1-spleen cell suspension in a large centrifuge tube, centrifuge at 1200 rpm for 8 min, discard the supernatant, and place the obtained cell pellet in a beaker containing 500 mL of sterile water and water bath at 37°C;

[0064] 4. Slowly add 1 mL of PEG dropwise to the cell pellet in the water bath within 90 s, let it stand for 90 s, and then slowly add 10 mL of RPMI-1640 medium dropwise within 5 min. After the addition is complete, centrifuge at 800 rpm for 6 min and discard the supernatant;

[0065] 5. Add 3 mL of RPMI-1640 medium to resuspend the cell pellet obtained in step 4, add the obtained resuspended solution to the thymocyte suspension obtained in step 1, blow it evenly with a dropper, and then evenly add the mixed cell suspension to a 96-well cell culture plate at about 100 μL per well;

[0066] 6. Place the cell culture plate in an incubator at 37°C for two weeks for cell fusion. After the culture, aspirate the cell supernatant in each well of the cell culture plate for detection to determine whether the cells in that well are positive. After detection, it is found that the cells in the well numbered 2D8 (hereinafter referred to as the "2D8 well") are positive hybridoma cells.

[0067] Step 6. Screening of fused cells, which specifically includes the following steps:

[0068] 1. Take the spleen of healthy Nile tilapia that has not been immunized, wash it in L15 medium, then grind, filter, and add the obtained tissue precipitate to percoll separation solution (4 mL of 52% percoll separation solution + 4 mL of 34% percoll separation solution), centrifuge at 500 g and 23°C for 30 min, and take the white annular layer cells suspended at the stratified layer. After washing with L15 medium, white blood cells are obtained.

[0069] 2. Resuspend the white blood cells with DMEM medium containing 10% FBS to obtain a white blood cell suspension, evenly distribute it into a 24-well cell culture plate at 100 μL / well, then add 2 μL of PHA to stimulate the cells. After culturing for 6 hours, blow up the cells, centrifuge and wash. The obtained white blood cell precipitate is resuspended with PBS (FACS Buffer) containing 2% FBS and added to a 96-well V-bottom plate.

[0070] 3. After centrifuging at 2500 rpm for 3 min, discard the supernatant, resuspend the cells with 100 μL of the cell supernatant in the 2D8 well, and add 100 μL of P3U1 cell supernatant to the negative control well to resuspend the cells. At the same time, incubate on ice for 30 min. After the incubation, centrifuge at 2500 rpm for 3 min, and then wash twice with 200 μL of FACS Buffer.

[0071] 4. After centrifugation and washing, resuspend the 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. After the incubation, centrifuge at 2500 rpm for 3 min, and wash twice with 200 μL of FACS Buffer.

[0072] 5. After washing, resuspend the cells with 200 μL of FACS Buffer, filter through a 200-mesh sieve, and analyze by flow cytometry. It is found that the antibody secreted by the positive hybridoma cells in the 2D8 well can bind to the surface protein of a certain cell population in Nile tilapia white blood cells.

[0073] Step 7. Cloning of positive hybridoma cells, which specifically includes the following steps:

[0074] 1. Take the thymus of healthy, non-immunized mice, wash, grind, centrifuge, and resuspend it with RPMI-1640 medium to obtain a thymocyte suspension. Centrifuge at 1000 rpm for 5 min, and resuspend and mix the resulting precipitate with 10 mL of RPMI-1640 medium containing 10% FBS.

[0075] 2. Resuspend the positive hybridoma cells in the 2D8 well, perform serial dilutions with RPMI-1640 medium containing 10% FBS, and count the diluted cells using a hemocytometer.

[0076] 3. Add approximately 100 of the diluted positive hybridoma cells to the thymocyte suspension that was resuspended and mixed again in step 1 above, mix well, and then evenly pipette into a 96-well cell culture plate, approximately 100 μL per well.

[0077] 4. Place the cell culture plate in a 37 °C incubator. After 10 - 14 days of culture, aspirate the cell supernatant. Take the spleen of non-immunized Nile tilapia, isolate the white blood cells, and stimulate the white blood cells with 2 μL of PHA for 6 h. Subsequently, place the stimulated white blood cells in a 96-well V-bottom plate, incubate with the cell supernatant in the 2D8 well, and finally perform detection by flow cytometry; as Figure 4 shown, after cloning the hybridoma cells in the 2D8 well, the antibody secreted by them can still stably bind to the surface protein of a certain cell population in Nile tilapia white blood cells. This cell population accounts for approximately 18.6% of the Nile tilapia spleen lymphocytes after 6 h of PHA stimulation. Further, the hybridoma cell line in the 2D8 well was named hybridoma cell line 2D8A7.

[0078] Furthermore, in this example, the antibody secreted by hybridoma cell line 2D8A7 was identified by semi-quantitative method, which includes the following steps:

[0079] 1. Obtain white blood cells according to the method in section 1 of step six, and then resuspend the white blood cells with FACS Buffer to obtain a white blood cell resuspension, which is loaded into a 2 mL EP tube.

[0080] 2. After centrifuging the above white blood cell resuspension at 2500 rpm for 3 min, discard the supernatant, resuspend the white blood cell pellet with 1.5 mL of the culture supernatant of hybridoma cell line 2D8A7 (containing the antibody secreted by hybridoma cell line 2D8A7), incubate on ice for 30 min, and wash the resulting white blood cell pellet twice with 1 mL of FACS Buffer.

[0081] 3. Resuspend the leukocyte pellet obtained after centrifugation with 1 mL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold, incubate on ice for 30 min in the dark, and wash the obtained pellet twice with 1 mL of FACS Buffer;

[0082] 4. Resuspend the pellet washed with FACS Buffer with 1 mL of FACS Buffer, and then perform flow sorting to collect the positive and negative cell populations respectively;

[0083] 5. Extract the total RNA of positive and negative cells by the Trizol method respectively, reverse transcribe to obtain cDNA templates, and amplify through PCR reactions to obtain the gene fragments of Nile tilapia β-actin, CD122, and CD3ε respectively. The primer sequences are shown in Table 1;

[0084] Table 1 Primer Information

[0085]

[0086]

[0087] 6. Add the PCR products (i.e., the gene fragments of Nile tilapia β-actin, CD122, and CD3ε) to 1% agarose gel for electrophoresis, adjust the loading amount of the PCR products by the band brightness to make the amount of β-actin in the positive and negative cell populations the same, and detect the gene expression levels after re-electrophoresis.

[0088] As Figure 5 shown, the expression levels of CD122 gene and CD3ε gene in the CD122 + cell population (i.e., the positive cell population) are extremely high, while CD122 - in the cell population (i.e., the negative cell population) hardly expresses CD122 and CD3ε, indicating that the positive cell population is indeed the CD122 + T lymphocyte population, the CD122 protein is mainly distributed on the surface of T cells, and it is proved that the antibody secreted by the hybridoma cell line 2D8A7 can specifically bind to the CD122 protein on the surface of Nile tilapia cells.

[0089] Furthermore, in this example, the binding situation between the antibody secreted by the hybridoma cell line 2D8A7 and the surface of tilapia lymphocytes was analyzed by immunofluorescence method, which includes the following steps:

[0090] 1. Obtain leukocytes according to the method in Section 1 of Step 6;

[0091] 2. Stimulate white blood cells with anti-Tilapia CD3ε monoclonal antibody and anti-Tilapia CD28 monoclonal antibody simultaneously for 6 h. After the stimulation, suspend the cells, wash them twice with PBS, then resuspend the cells with PBS, evenly drop the cell suspension on a glass slide, place the glass slide in a humid box, let it stand for 1 h, and then fix it in methanol for 5 min to prepare a leukocyte smear of Nile tilapia. Among them, CD3ε and CD28 are two important targets for T cell activation. Using CD3ε and CD28 antibodies in combination to stimulate T cells in vitro can simulate the dual-signal effect of T cell activation in vivo.

[0092] 3. After drying the methanol on the surface of the leukocyte smear, draw a hydrophobic circle around the cell circle with a hydrophobic pen, and then sequentially drop 100 μL of 1% BSA blocking solution dissolved in PBS, 100 μL of the culture supernatant of hybridoma cell line 2D8A7 (containing the antibody secreted by hybridoma cell line 2D8A7), and 100 μL of anti-mouse Alexa Fluor594 fluorescent secondary antibody diluted 400 times on the cells. After all the dropping operations are completed, place the leukocyte smear in an incubator at 37 °C for 1 h. After the incubation, wash it twice with PBST and once with PBS, and the washing time for each time is 5 min.

[0093] 4. Drop 2 μL of DAPI nuclear dye on the leukocyte smear, cover it with a coverslip, and observe it under a fluorescence microscope.

[0094] As Figure 6 shown (the "CD122" part shows the staining effect of CD122 protein on the cell surface, and the "DAPI" part shows the staining effect of the cell nucleus). Some white blood cells (i.e., the cells shown in the "Merge" part, and the image of the "Merge" part is obtained by superimposing the "CD122" part and the "DAPI" part) show a red positive reaction in a circle under the fluorescence microscope, indicating that the antibody secreted by hybridoma cell line 2D8A7 can specifically bind to CD122 protein on the surface of Nile tilapia lymphocytes, and after activating T cells with CD3ε and CD28 monoclonal antibodies, the expression level of CD122 protein is up-regulated.

[0095] In this example, using the retrovirus attack method, the Nile tilapia CD122 gene fragment was ligated to the plasmid pMSCV, and then it was transfected into 293T cells by calcium transfection to prepare retrovirus, and the obtained virus was used to infect mouse homologous NIH / 3T3 cells, so that CD122 protein was successfully expressed on the surface of mouse NIH / 3T3 cells, overcoming the problem that it is difficult to obtain Nile tilapia CD122 protein in its natural state due to the complex transmembrane structure of the protein itself.

[0096] Furthermore, after fusing the spleen cells of immunized mice with hybridoma cells, cells capable of secreting antibodies were screened out by flow cytometry, and the cells in the positive wells were cloned. It was determined by flow screening, semi-quantitative screening, and immunofluorescence screening that the antibody produced by the hybridoma cell line 2D8A7 could specifically bind to the CD122 protein on the surface of Nile tilapia lymphocytes and could specifically recognize Nile tilapia CD122 + cells.

[0097] Example 2:

[0098] This example provides a monoclonal antibody against tilapia CD122 protein (i.e., "anti-tilapia CD122 protein monoclonal antibody") secreted by the hybridoma cell line 2D8A7 obtained in Example 1, and the preparation process of the monoclonal antibody includes the following steps:

[0099] Ascites preparation: Take BALB / c mice at 8 - 10 weeks of age, and inject 450 μL of sterile paraffin oil into the abdominal cavity of each mouse; 10 days later, take the hybridoma cell line 2D8A7 with good growth state, wash away the culture medium with PBS, resuspend the cells with 350 μL of sterile PBS, and inject 2×10 5 cells into the abdominal cavity of each mouse; Observe the immunized mice every day. When the abdomen of the mouse bulges and it has difficulty moving, decapitate and sacrifice the immunized mouse, and collect ascites; Centrifuge the ascites at 2000 rpm for 5 min, take the pale yellow ascites in the middle, aliquot and store it at -80 °C;

[0100] Antibody purification: Take 250 μL of rProtein G Agarose in a 15 mL centrifuge tube, wash it three times with PBS, then add 450 μL of the aliquoted and stored ascites and dilute it to 8 mL with PBS, and incubate overnight at 4 °C; After washing the beads 6 times with PBS, elute with 500 μL of 0.1 M Glycine-HCl (pH = 2.8) to obtain the monoclonal antibody against tilapia T lymphocyte cytokine IL-2, and aliquot and store it at -80 °C after adding 1 / 10 volume of 1 M Tris-HCl (pH = 8.5) to neutralize the pH value.

[0101] Example 3:

[0102] This example provides an application of the monoclonal antibody described in Example 2 in the study of adaptive immune response in teleost fish, which specifically includes the following content:

[0103] I. Application of anti-tilapia CD122 protein monoclonal antibody in response to Edwardsiella piscicida infection, which includes the following steps:

[0104] 1. Culture Edwardsiella piscicida in TYB medium containing Col resistance for 8 hours, centrifuge and wash, adjust OD 600 = 1 on an enzyme-linked immunosorbent assay (ELISA) reader, and then dilute 40-fold with PBS;

[0105] 2. Take several healthy Nile tilapia, divide them into an experimental group and a control group. In the experimental group, intraperitoneally inject 200 μL of the diluted Edwardsiella piscicida into each tilapia, and inject the same dose of PBS into the control group;

[0106] 3. On the 5th day after infection, sacrifice the Nile tilapia in the experimental group and the control group, take out the spleen, isolate white blood cells by density gradient centrifugation (i.e., the method in Section 1 of Step 6 above), resuspend them in FACS Buffer, add the white blood cell suspension to a 96-well V-bottom plate, centrifuge at 2500 rpm for 3 min, discard the supernatant, resuspend the cell pellet with 100 μL of the anti-Nile tilapia CD122 protein monoclonal antibody in Example 2, incubate on ice for 30 min, centrifuge the cells and wash twice with 200 μL of FACS Buffer; then 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, centrifuge the cells and wash twice with 200 μL of FACS Buffer; then resuspend the cells with 200 μL of FACS Buffer, filter through a 200-mesh sieve and perform flow cytometry.

[0107] As Figure 7 shown, by flow cytometry analysis, 5 days after infection with Edwardsiella piscicida, the proportion of CD122 + cells in T cells increased from 10.8% before infection to 30.2% after infection (as shown in the "E. piscicida day 5" part), indicating that the proportion of T cells expressing CD122 + in Nile tilapia increased sharply in response to pathogen infection and played a role in the antibacterial immune process.

[0108] II. Application of the anti-Nile tilapia CD122 protein monoclonal antibody in detecting the distribution of CD122 protein in Nile tilapia immune-related tissues, which specifically includes the following steps:

[0109] 1. Take healthy Nile tilapia, and collect peripheral blood, head kidney, liver and spleen respectively, and isolate white blood cells from each tissue. When isolating peripheral blood white blood cells, first use a syringe to aspirate 2 mL of anticoagulant (15 mM sodium citrate, 450 mM NaCl, 0.1 M glucose, 10 mM EDTA, pH 7.0), 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 separation solution;

[0110] 2. Resuspend the white blood cells of each tissue with 1 mL of FACS buffer respectively, add them to a 96-well V-bottom plate, centrifuge at 2500 rpm for 3 min, and discard the supernatant; Suspend the white blood cells of each tissue with 100 μL of the anti-tilapia CD122 monoclonal antibody in Example 2 respectively, incubate on ice for 30 min, centrifuge the white blood cells of each tissue, and wash twice with 200 μL of FACS Buffer; Suspend the centrifuged cells with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000-fold respectively, and incubate on ice for 30 min in the dark, centrifuge the cells, and wash twice with 200 μL of FACS Buffer; After suspending the cells with 200 μL of FACS buffer, perform flow cytometry detection.

[0111] As Figure 8 shown, the proportions of CD122+ lymphocytes in peripheral blood (PBL), head kidney (HK), liver (Liver) and spleen (SP) lymphocytes are 9.13%, 7.65%, 22.9% and 7.55% respectively, indicating that the anti-tilapia CD122 protein monoclonal antibody in Example 2 can activate T lymphocytes in multiple tissues.

[0112] III. Application of the anti-tilapia CD122 protein monoclonal antibody in specifically recognizing tilapia CD122 protein and increasing the expression of tilapia CD122 protein, which includes the following steps:

[0113] 1. Take the spleen of unimmunized healthy Nile tilapia, and obtain spleen lymphocytes using density gradient centrifugation; Stimulate spleen lymphocytes with PHA in vitro for 6 h and 12 h respectively, and stimulate spleen lymphocytes with anti-tilapia CD3ε monoclonal antibody and anti-tilapia CD28 monoclonal antibody simultaneously for 6 h and 12 h; At the same time, prepare Nile tilapia infected with Edwardsiella piscicida for 3 and 5 days, take out their spleens respectively, grind and centrifuge to obtain spleen lymphocytes infected for 3 days and spleen lymphocytes infected for 5 days respectively;

[0114] 2. Prepare NP40 lysis buffer. Lyse all the six kinds of spleen lymphocytes obtained with NP40 lysis buffer on ice for 30 min. After the lysis, centrifuge at 12,000 rpm and 4 °C for 10 min. Take the supernatant and place it in a 1.5 mL EP tube, and mix it with 5× loading buffer. Boil it in a boiling water bath for 10 minutes to obtain protein samples corresponding to the six kinds of spleen lymphocytes.

[0115] 3. Add each protein sample into the wells of the SDS PAGE gel and conduct gel electrophoresis by applying an electric current.

[0116] 4. Take out the gel from the gel plate, remove the stacking gel, closely attach the separating gel to the NC membrane, expel the air bubbles, set the voltage at 100 V, place the membrane transfer instrument in an ice-water bath, and transfer all the proteins on the gel to the NC membrane with the membrane transfer instrument.

[0117] After the membrane transfer, prepare 15 mL of skim milk powder with PBST (PBS + 0.5% Tween 20). Place the NC membrane in the skim milk powder and block it at room temperature on a shaker for one hour, and then wash it three times with PBST, 10 minutes each time.

[0118] 4. Place the washed membrane in the culture supernatant of the hybridoma cell line 2D8A7 (containing the antibody secreted by the hybridoma cell line 2D8A7), and incubate it overnight on a shaker at 4 °C, ensuring that the membrane is completely covered by the supernatant during incubation.

[0119] After the incubation, wash the membrane three times with PBST, 10 minutes each time. After that, transfer the membrane to a skim milk powder solution containing anti-mouse Alexa Fluor 680 (Abcam) and incubate it for one hour. After the incubation, wash it three times with PBST, 10 minutes each time. Scan the NC membrane with an Odyssey CLX imaging system.

[0120] As Figure 9 shown, the monoclonal antibody against tilapia CD122 protein can specifically bind to Nile tilapia CD122 protein. The size of the CD122 protein is about 70 Kd, which is the same as the predicted protein size. And after being stimulated in vitro with PHA, anti-tilapia CD3ε monoclonal antibody + anti-tilapia CD28 monoclonal antibody (i.e., Figure 9 "αCD3ε / 28" in

[0121] Example 4:

[0122] This embodiment provides an application of the anti-rohu CD122 monoclonal antibody in Example 2 in the preparation of fish disease prevention and treatment reagents / drugs.

[0123] In summary, the present invention uses the lentivirus infection method to transfer the plasmid containing the gene fragment of Nile tilapia CD122 into the mouse homologous NIH / 3T3 cell line, enabling the cell surface to express active tilapia CD122 protein. Moreover, a screening and identification mechanism combining flow cytometry, semi-quantitative detection, and immunofluorescence technology is constructed, and a monoclonal antibody against tilapia CD122 is successfully prepared. The relevant experimental results provide strong evidence for understanding CD122 as a marker molecule for early vertebrate T cell activation, also provide important tools and technical support for the study of fish adaptive immunity, especially the study of fish T cell immunity, and at the same time provide a reference basis for fish disease prevention and control and vaccine efficacy evaluation.

[0124] 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 within the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting anti-tilapia CD122 protein monoclonal antibody, characterized in that: The hybridoma cell line is hybridoma cell line 2D8A7, which was deposited in the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with the deposit number CCTCC NO: C2024142, and the deposit address is Wuhan University, Wuhan, China.

2. A method for preparing the hybridoma cell line 2D8A7 according to claim 1, characterized in that: The steps include: Construction of the gene expression vector for tilapia T lymphocyte surface membrane protein CD122; Retroviral preparation; NIH / 3T3 cells were infected by retrovirus; NIH / 3T3 cells infected with retrovirus were used as antigens for animal immunization; Using immunized animals for cell fusion to obtain positive hybridoma cells; and positive hybridoma cell clones.

3. A monoclonal antibody against tilapia CD122 protein secreted by the hybridoma cell line 2D8A7 according to claim 1.

4. A method for preparing the monoclonal antibody according to claim 3, characterized in that: The steps include: Ascites preparation: mice were given intraperitoneal injection of sterile paraffin oil; Resuspend the hybridoma cell line 2D8A7 according to claim 1, and then inject it intraperitoneally into mice; The mice were killed, and the ascites was collected and centrifuged and stored in aliquots; The ascites is purified to obtain the monoclonal antibody against the tilapia CD122 protein.

5. Use of the monoclonal antibody according to claim 3 in response to Edwardsiella piscicida infection.

6. Use of the monoclonal antibody according to claim 3 in detecting the distribution of CD122 protein in tilapia immune-related tissues.

7. The use according to claim 6, characterized in that The immune-related tissues include one or more of peripheral blood, head kidney, liver and spleen.

8. Use of the monoclonal antibody according to claim 3 in specifically recognizing tilapia CD122 protein.

9. Use of the monoclonal antibody according to claim 3 in improving the expression of tilapia CD122 protein.

10. Use of the monoclonal antibody according to claim 3 in the preparation of fish disease prevention and treatment reagents / medicines.

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