Hybridoma cell strain secreting anti-rolof fish cd122 protein monoclonal antibody and application thereof
By expressing tilapia CD122 protein in mouse NIH/3T3 cells using lentiviral infection, a hybridoma cell line 2D8A7 that secretes antibodies was prepared, solving the problem of the difficulty in preparing CD122 protein and providing tool support for tilapia T-cell immunity research and disease control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA NORMAL UNIV
- Filing Date
- 2025-02-13
- Publication Date
- 2026-06-16
AI Technical Summary
Existing technologies make it difficult to express CD122 protein with complete natural structure and function in large quantities in vitro, which makes it difficult to prepare tilapia CD122 protein monoclonal antibodies, affecting in-depth research on T cell adaptive immunity and disease control in tilapia.
The Nile tilapia CD122 gene fragment was transfected into mouse NIH/3T3 cells using a lentiviral infection method to construct a retroviral vector. Through cell fusion and screening, a hybridoma cell line 2D8A7 secreting anti-tilapia CD122 protein was obtained, and the corresponding monoclonal antibody was prepared.
A monoclonal antibody that can specifically recognize and bind to tilapia CD122 protein was successfully prepared, providing a reference for research on fish adaptive immunity and disease control, activating T cells and responding to pathogen infection, and increasing CD122 protein expression.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fish immunology, specifically relating to a hybridoma cell line that secretes a monoclonal antibody against tilapia CD122 protein, the monoclonal antibody, its preparation method, and its application. Background Technology
[0002] During T cell activation and proliferation, CD122 is the β subunit of the interleukin-2 / interleukin-15 (IL-2 / IL-15) receptor, mainly located 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 through the intracellular domain of CD122. Tyrosine phosphorylation of the CD122 intracellular domain can serve as a docking site for STAT5, thereby activating JAK-STAT signal transduction and participating in various biological functions such as T cell activation, proliferation, and cytotoxicity. Therefore, CD122 is often considered a marker receptor for T cell activation and on the surface of CD8+ T cells.
[0003] Due to the complexity of transmembrane protein structures, it remains difficult to express CD122 protein with its complete natural structure and original function in large quantities in vitro. This makes it difficult to apply traditional monoclonal antibody preparation methods to the preparation of monoclonal antibodies against CD122 protein on the membrane surface. Furthermore, the difficulty in preparing specific monoclonal antibodies against tilapia CD122 protein hinders in-depth research into the processes and mechanisms of T lymphocyte activation and cytotoxicity in tilapia adaptive immunity. Summary of the Invention
[0004] To address the deficiencies in the prior art, this invention provides a hybridoma cell line that secretes a monoclonal antibody against tilapia CD122 protein and its application. This application can express active tilapia CD122 protein on the cell surface and successfully prepares a monoclonal antibody against tilapia CD122, providing a reference for research on fish adaptive immunity, fish disease control, and vaccine efficacy evaluation.
[0005] The technical solution provided by this invention to solve the above-mentioned technical problems is as follows:
[0006] On the one hand, a hybridoma cell line 2D8A7 that secretes a monoclonal antibody 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 accession number CCTCC NO: C2024142, and the deposit address is Wuhan University, Wuhan, China.
[0007] On the other hand, a method for preparing the above-mentioned hybridoma cell line 2D8A7 is also provided, which includes the following steps:
[0008] Construction of an expression vector for the CD122 gene, a surface membrane protein of tilapia T lymphocytes;
[0009] Preparation of retroviruses;
[0010] NIH / 3T3 cells were infected with retroviruses;
[0011] NIH / 3T3 cells infected with retroviruses were used as antigens for animal immunization;
[0012] Cell fusion was performed using immunized animals to obtain positive hybridoma cells;
[0013] And positive hybridoma cell clones.
[0014] On the other hand, a monoclonal antibody against tilapia CD122 protein secreted by the aforementioned 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 includes the following steps:
[0016] Ascites preparation: Mice were taken and injected intraperitoneally with sterile paraffin oil;
[0017] The above hybridoma cell line 2D8A7 was resuspended and then injected intraperitoneally into mice.
[0018] The mice were euthanized, ascites fluid was collected, centrifuged, and aliquoted for storage.
[0019] The ascites fluid was purified to obtain the monoclonal antibody against the tilapia CD122 protein.
[0020] On the other hand, the application of the above-mentioned monoclonal antibody in response to Edwardsiella piscicida infection 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 the specific recognition of tilapia CD122 protein is also provided.
[0024] On the other hand, the application of the above-mentioned monoclonal antibody in enhancing the expression of CD122 protein in tilapia is also provided.
[0025] On the other hand, the application of the above-mentioned monoclonal antibody in the preparation of fish disease prevention and control reagents / drugs is also provided.
[0026] Preferably, the fish disease prevention and control reagent / drug includes a fish disease prevention and control vaccine.
[0027] This invention employs a lentiviral infection method to transform a plasmid containing the Nile tilapia CD122 gene fragment into a mouse homologous NIH / 3T3 cell line, enabling the expression of active tilapia CD122 protein on the cell surface. Furthermore, a screening and identification mechanism combining flow cytometry, semi-quantitative detection, and immunofluorescence techniques was constructed, successfully producing a monoclonal antibody against tilapia CD122. This provides strong evidence for understanding CD122 as a marker molecule of early vertebrate T cell activation and also offers a reference for research on fish adaptive immunity, fish disease control, and vaccine efficacy evaluation. Attached Figure Description
[0028] Figure 1 Stained images of 293T cells;
[0029] Figure 2 Stained images of NIH / 3T3 cells;
[0030] Figure 3 Flow cytometry analysis results of NIH / 3T3 cells infected with retroviruses;
[0031] Figure 4 The results of flow cytometry analysis of hybridoma cell line 2D8A7 combined with specific cell populations in leukocytes are shown, where (a) is a histogram and (b) is a density plot.
[0032] Figure 5 Electrophoresis images of each gene in CD122-positive and CD122-negative cell populations;
[0033] Figure 6 A staining pattern showing the binding of antibodies secreted by hybridoma cell line 2D8A7 to CD122 protein;
[0034] Figure 7 The flow cytometry results of the anti-tilapia CD122 monoclonal antibody detection group and the control group were shown in (a) as histogram and (b) as density plot.
[0035] Figure 8 Flow cytometry analysis results of lymphocytes from head kidney, peripheral blood, liver, and spleen for detection of anti-tilapia CD122 monoclonal antibody;
[0036] Figure 9 Electrophoresis diagram for Western blot detection of the binding of anti-tilapia CD122 monoclonal antibody to specific proteins in lymphocytes; Detailed Implementation
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0038] Example 1:
[0039] This embodiment provides a hybridoma cell line 2D8A7 that secretes an antibody against tilapia CD122 protein. The hybridoma cell line 2D8A7 was deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with accession number CCTCC NO: C2024142, and the deposit address is Wuhan University, Wuhan, China. The tilapia is Nile tilapia (Oreochromis niloticus).
[0040] Furthermore, the method for preparing the above-mentioned hybridoma cell line 2D8A7 includes the following steps:
[0041] Step 1: Construction of the expression vector for the CD122 gene, a surface membrane protein of tilapia T lymphocytes, which includes the following steps:
[0042] 1. Obtain the CD122 gene fragment sequence of Nile tilapia (accession number: XM_019360337.2) from the NCBI database. The gene fragment was cloned by Sangon Biotech Co., Ltd., and the cloned CD122 gene fragment was ligated into the pMSCV vector using XhoⅠ and EcoRI as restriction enzyme sites.
[0043] 2. The pMSCV vector containing the CD122 gene fragment was introduced into the glycerol bacterium strain, and the glycerol bacterium strain was then cultured in 30 mL of LB liquid medium containing ampicillin resistance. The retroviral expression vector containing the CD122 gene fragment, namely the pMSCV-CD122 plasmid, was then extracted from the culture medium.
[0044] Step 2, retrovirus preparation, specifically includes the following steps:
[0045] 1. Resuscitate 293T cells in DMEM complete medium (DMEM medium + 10% FBS + 1% double antibiotics, by weight) and culture them in 6cm diameter culture dishes. After the cells have filled the culture dish, passage them.
[0046] During passage, the cell culture waste liquid in the culture dish was aspirated, the remaining cells were washed with 3 mL PBS, the PBS was discarded, 1 mL trypsin was added and shaken well, and the cells were digested in a 37°C incubator for 30 seconds. The trypsin was discarded, and the cells were then blown off and resuspended with 3 mL DMEM medium, and then transferred to a 10 cm culture dish for culture.
[0047] After three passages, 293T cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in DMEM medium. Cell counts were performed using a hemocytometer, and then 2.2 × 10⁶ cells were seeded onto 6 cm diameter culture dishes. 6 One cell was added to a 3 mL DMEM medium and cultured.
[0048] 2. After 10 hours of incubation, perform the transfection experiment, which includes the following:
[0049] 2.1 Prepare the transfection system solution, which includes 10 μg pMSCV-CD122 plasmid, 2 μg pCMV-VSV-G, 5 μg pCL-Eco plasmid, 50 μL 2.5M CaCl2 solution, and finally add sterile water to make up to 500 μL. Then, gently blow up the mixture with a dropper and slowly add 500 μL 2×HEBS buffer.
[0050] 2.2 Discard the DMEM medium used for cell culture after three passages, add 3 mL of DMEM complete medium containing 25 μM chloroquine, then add 1 mL of the above transfection solution, and incubate at 37°C for 6-8 hours for transfection. Figure 1 As shown, 293T cells transfected with this virus exhibited green fluorescence under a fluorescence microscope.
[0051] 2.3 After culturing for 6-8 hours, discard the supernatant and replace it with 3 mL of DMEM complete medium. Subsequently, replace the DMEM complete medium every 12 hours, with each replacement using 3-4 mL of DMEM complete medium. After 48 hours of cell culture, collect the supernatant, centrifuge it at 1200 rpm for 8 minutes, collect the supernatant again, aliquot it, and freeze it at -80°C. The supernatant collected again is the retrovirus suspension.
[0052] Step 3: NIH / 3T3 cell infection, which specifically includes the following steps:
[0053] 1. Resuscitate NIH / 3T3 cells and culture them in 6cm culture dishes, passaged three times (the passage method can be the same as described in Section 1 of Step 2). After passage, wash the cells with PBS, add 1mL of trypsin and digest at 37℃ for 30s. Then, blow off all adherent cells with DMEM medium and resuspend them. Transfer the resuspended solution to a 15mL centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, and retain the cell pellet. Resuspend the cell pellet in DMEM medium, blow the cells evenly, count them using a hemocytometer, and then seed 2×10⁶ cells into 10cm diameter culture dishes. 5 One NIH / 3T3 cell was added to 8 mL of DMEM medium for cell culture.
[0054] 2. After culturing for 12 hours, discard the DMEM medium, slowly add 6 mL of DMEM complete medium containing 5 μM polybrene to adhere the cells, and then slowly add 600 μL of the above retrovirus suspension to the NIH / 3T3 cells. Gently shake to mix, and incubate at 37°C.
[0055] 3. After culturing for 6 hours, discard the supernatant and add 8 mL of preheated DMEM complete medium. Then replace the DMEM complete medium every 12 hours for a total of 3 times, and each time the volume of DMEM complete medium is 8-10 mL to ensure that the polyaluminamine is washed away.
[0056] After culturing for 48 hours, the supernatant was discarded, and the cell pellet was retained. The cell pellet was digested with trypsin for 1 minute, and then resuspended in 3 mL of LDM medium. The resulting resuspended solution was added to a 15 mL centrifuge tube, and PBS was added to bring the volume to 10 mL. The tube was centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cell pellet was retained. The cell pellet was then resuspended in 10 mL of PBS, and centrifuged again at 1200 rpm for 3 min. The supernatant was discarded, and the cell pellet was retained. The cell pellet obtained from the last centrifugation was then resuspended in 200 μL of PBS.
[0057] like Figure 2 As shown, fluorescence microscopy revealed that NIH / 3T3 cells exhibited green fluorescence, confirming that the retrovirus containing the Nile tilapia CD122 gene successfully infected NIH / 3T3 cells. Figure 3 As shown, flow cytometry analysis revealed that NIH / 3T3 cells had a high infection efficiency, reaching 56.9%.
[0058] Step four, animal immunization, specifically includes the following steps:
[0059] 6-week-old BALB / c mice were immunized with retroviral-infected NIH / 3T3 cells as antigen via intraperitoneal injection. A total of four immunizations were administered, with the second immunization given two weeks after the initial immunization, followed by immunizations every week. Each mouse received approximately 1.5 × 10¹² cells per injection. 7 One NIH / 3T3 cell.
[0060] Step 5, cell fusion, specifically includes the following steps:
[0061] 1. Five days after the fourth immunization, the thymus of unimmunized 6-week-old healthy mice was taken, washed, ground, and centrifuged with RPMI-1640 medium, and then resuspended in HAT-RPMI-1640 medium. Large pieces of tissue were discarded, and the thymus cell suspension was obtained and placed in a 37°C incubator for subsequent experiments. The spleen of mice immunized with the above animals was also taken, washed, ground, and centrifuged with RPMI-1640 medium, and then the spleen cells were resuspended in RPMI-1640 medium to obtain a spleen cell suspension.
[0062] 2. One week before fusion, resuscitate P3-X63-Ag8U1 (P3U1) cells. Two days before fusion, take P3U1 cells of appropriate density and passage them into culture flasks. Change the culture medium 12 hours before fusion.
[0063] 3. The above-mentioned spleen cell suspension was fused with P3U1 cells that had been resuscitated and passaged to obtain P3U1-spleen cell suspension. The P3U1-spleen cell suspension was placed in a large centrifuge tube and centrifuged at 1200 rpm for 8 min. The supernatant was discarded, and the resulting cell pellet was placed in a beaker containing 500 mL of sterile water and incubated in a water bath at 37°C.
[0064] 4. Slowly add 1 mL of PEG to the cell pellet in the water bath within 90 seconds, let stand for 90 seconds, and then slowly add 10 mL of RPMI-1640 medium over 5 minutes. After the addition is complete, centrifuge at 800 rpm for 6 minutes and discard the supernatant.
[0065] 5. Add 3 mL of RPMI-1640 medium to the cell pellet obtained in step 4 to resuspend the cell pellet. Add the resulting resuspended solution to the thymocyte suspension obtained in step 1, mix well with a dropper, and then add approximately 100 μL of the mixed cell suspension evenly to each well of a 96-well cell culture plate.
[0066] 6. The cell culture plate was placed in a 37°C incubator for two weeks to allow for cell fusion. After the culture was completed, the cell supernatant from each well of the cell culture plate was aspirated for testing to determine whether the cells in that well were positive. The test revealed that the cells in well numbered 2D8 (hereinafter referred to as "well 2D8") were positive hybridoma cells.
[0067] Step 6, fusion cell screening, specifically includes the following steps:
[0068] 1. Take the spleen of healthy Nile tilapia that has not been immunized, wash it in L15 medium, grind and filter it, add the resulting tissue precipitate to Percoll separation solution (4 mL 52% Percoll separation solution + 4 mL 34% Percoll separation solution), centrifuge at 500g, 23℃ for 30 min, take the white ring layer cells suspended at the separation point, wash them with L15 medium to obtain white blood cells;
[0069] 2. Resuspend leukocytes in DMEM medium containing 10% FBS to obtain a leukocyte suspension. Distribute the suspension evenly to 24-well cell culture plates at a rate of 100 μL / well. Then add 2 μL of LPHA to stimulate the cells. After culturing for 6 hours, blow up the cells, centrifuge and wash them. Resuspend the resulting leukocyte pellet in PBS (FACS Buffer) containing 2% FBS and add it to 96-well V-type plates.
[0070] 3. After centrifuging at 2500 rpm for 3 min, discard the supernatant and resuspend the cells with 100 μL of cell supernatant in 2D8 wells. Add 100 μL of LP3U1 cell supernatant to the negative control wells to resuspend the cells. Incubate on ice for 30 min. After incubation, centrifuge at 2500 rpm for 3 min and wash twice with 200 μL of LFACS Buffer.
[0071] 4. After centrifugation and washing, the cells were suspended with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000 times. The cells were incubated on ice for 30 min in the dark. After incubation, the cells were centrifuged at 2500 rpm for 3 min and washed twice with 200 μL of LACS Buffer.
[0072] 5. After washing, the cells were suspended in 200 μL of FACS Buffer, filtered through a 200-mesh sieve, and analyzed by flow cytometry. It was found that the antibodies secreted by the positive hybridoma cells in the 2D8 wells could bind to the surface proteins of a certain cell group in Nile tilapia leukocytes.
[0073] Step 7: Positive hybridoma cell cloning, which specifically includes the following steps:
[0074] 1. Take the thymus of healthy mice that have not been immunized, wash, grind, centrifuge and resuspend in RPMI-1640 medium to obtain thymocyte suspension, centrifuge at 1000 rpm for 5 min, and resuspend the resulting precipitate in 10 mL of RPMI-1640 medium containing 10% FBS and mix well.
[0075] 2. Blow up the positive hybridoma cells from the above 2D 8 wells and serially dilute them with RPMI-1640 medium containing 10% FBS. Count the cells using a hemocytometer after dilution.
[0076] 3. Take about 100 diluted positive hybridoma cells and add them to the thymocyte suspension in step 1 above. Mix well and then add evenly to a 96-well cell culture plate, about 100 μL per well.
[0077] 4. Place the cell culture plate in a 37℃ incubator and culture for 10-14 days, then aspirate the cell supernatant. Collect the spleen from unimmunized Nile tilapia and isolate leukocytes, stimulating them with 2 μL PHA for 6 hours. Subsequently, place the stimulated leukocytes in 96-well V-plates and incubate with cell supernatant from 2D8 wells. Finally, analyze the results by flow cytometry. Figure 4 As shown, after cloning, the hybridoma cells in the 2D8 wells still have antibodies that can stably bind to the surface proteins of a certain cell group in Nile tilapia leukocytes. This cell group accounts for about 18.6% of the Nile tilapia spleen lymphocytes after 6 hours of PHA stimulation. The hybridoma cell line in the 2D8 wells is further named hybridoma cell line 2D8A7.
[0078] Furthermore, this embodiment uses a semi-quantitative method to identify antibodies secreted by hybridoma cell line 2D8A7, which includes the following steps:
[0079] 1. Obtain white blood cells according to the method in Section 1 of Step 6, then resuspend the white blood cells with FACS Buffer to obtain a white blood cell resuspension, and put it into a 2mL EP tube;
[0080] 2. After centrifuging the above leukocyte resuspension at 2500 rpm for 3 min, discard the supernatant, and resuspend the leukocyte pellet with 1.5 mL of culture supernatant of hybridoma cell line 2D8A7 (containing antibodies secreted by hybridoma cell line 2D8A7). Incubate on ice for 30 min, and wash the leukocyte pellet obtained after centrifugation twice with 1 mL of LFACS Buffer.
[0081] 3. Resuspend the leukocyte pellet obtained after centrifugation with 1 mL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000 times. Incubate on ice for 30 min under light-protected conditions. Wash the pellet obtained after centrifugation twice with 1 mL of LFACS Buffer.
[0082] 4. Resuspend the precipitate after washing with FACS Buffer using 1 mL of FACS Buffer, and then perform flow cytometry to collect positive and negative cell populations separately;
[0083] 5. Total RNA was extracted from positive and negative cells using the Trizol method, and cDNA templates were obtained by reverse transcription. The cDNA templates were then amplified by PCR to obtain gene fragments of β-actin, CD122, and CD3ε from Nile tilapia. The primer sequences are shown in Table 1.
[0084] Table 1 Primer Information
[0085]
[0086]
[0087] 6. The PCR products (i.e. gene fragments of Nile tilapia β-actin, CD122, and CD3ε) were added to a 1% agarose gel for electrophoresis. The amount of PCR products loaded was adjusted by the band brightness to ensure that the amount of β-actin in the positive and negative cell populations was the same. After electrophoresis, the expression level of each gene was detected.
[0088] like Figure 5 As shown, CD122 + The expression levels of both CD122 and CD3ε genes were extremely high in the cell population (i.e., the positive cell population), while CD122... - The cell population (i.e., the negative cell population) basically does not express CD122 or CD3ε, indicating that the positive cell population is indeed CD122. + The CD122 protein is mainly distributed on the surface of T lymphocytes, and it has been demonstrated 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 embodiment, the binding of antibodies secreted by hybridoma cell line 2D8A7 to the surface of tilapia lymphocytes was analyzed by immunofluorescence, which included the following steps:
[0090] 1. Obtain white blood cells according to the method in Section 1 of Step Six;
[0091] 2. Leukocytes were simultaneously stimulated for 6 hours with anti-tilapia CD3ε monoclonal antibody and anti-tilapia CD28 monoclonal antibody. After stimulation, the cells were suspended, washed twice with PBS, and then resuspended with PBS. The cell suspension was then evenly dropped onto a glass slide, which was placed in a humidified chamber and allowed to stand for 1 hour. Finally, the slide was fixed in methanol for 5 minutes to prepare Nile tilapia leukocyte droplets. CD3ε and CD28 are two important targets for T cell activation. The combined use of CD3ε and CD28 antibodies to stimulate T cells in vitro can simulate the dual signaling action of T cell activation in vivo.
[0092] 3. After drying the surface of the leukocyte droplet with methanol, draw a hydrophobic circle around the cell ring with a hydrophobic pen. Then, add 100 μL of 1% BSA blocking solution dissolved in PBS, 100 μL of culture supernatant of hybridoma cell line 2D8A7 (containing antibodies secreted by hybridoma cell line 2D8A7), and 100 μL of anti-mouse Alexa Fluor594 fluorescent secondary antibody diluted 400 times to the cells in sequence. After all the addition operations are completed, incubate the leukocyte droplet in a 37°C incubator for 1 hour. After incubation, wash twice with PBST and once with PBS, with each washing time being 5 minutes.
[0093] 4. Add 2 μL of DAPI nuclear dye to the leukocyte droplet, cover with a coverslip, and observe under a fluorescence microscope.
[0094] like Figure 6 As shown (the “CD122” section represents the staining effect of CD122 protein on the cell surface, and the “DAPI” section represents the staining effect of the cell nucleus), some leukocytes (i.e., the cells shown in the “Merge” section, the “CD122” section and the “DAPI” section are superimposed to form the “Merge” image) show a red positive reaction under a fluorescence microscope, indicating that the antibody secreted by the hybridoma cell line 2D8A7 can specifically bind to the CD122 protein on the surface of Nile tilapia lymphocytes, and the expression level of CD122 protein is upregulated after T cells are activated by stimulation with CD3ε and CD28 monoclonal antibodies.
[0095] In this embodiment, the Nile tilapia CD122 gene fragment was ligated to the plasmid pMSCV using a retroviral attack method. Subsequently, the plasmid was transfected into 293T cells using a calcium transfer method to prepare a retrovirus. The resulting virus was then used to infect mouse homologous line NIH / 3T3 cells, successfully expressing the CD122 protein on the surface of mouse NIH / 3T3 cells. This overcame the difficulty in obtaining the naturally occurring tilapia CD122 protein due to the complex transmembrane structure of the protein itself.
[0096] Furthermore, after fusing spleen cells from immunized mice with hybridoma cells, flow cytometry was used to screen for antibody-secreting cells, and positive well cells were cloned. Flow cytometry, semi-quantitative screening, and immunofluorescence screening confirmed that the antibody produced by hybridoma cell line 2D8A7 specifically binds to the CD122 protein on the surface of Nile tilapia lymphocytes, and specifically recognizes Nile tilapia CD122. + cell.
[0097] Example 2:
[0098] This embodiment provides a monoclonal antibody against tilapia CD122 protein secreted by the hybridoma cell line 2D8A7 obtained in Example 1 (i.e., "anti-tilapia CD122 protein monoclonal antibody"), and the preparation process of the monoclonal antibody includes the following steps:
[0099] Ascites preparation: 8-10 week old BALB / c mice were intraperitoneally injected with 450 μL of sterile paraffin oil. Ten days later, the hybridoma cell line 2D8A7, in good growth condition, was collected. The culture medium was washed off with PBS, and the cells were resuspended in 350 μL of sterile PBS. Each mouse was then intraperitoneally injected with 2 × 10⁻⁶ cells. 5 Cells; observe the immunized mice daily, and when the mice's abdomens swell and they become unable to move easily, euthanize them by dislocating their cervical vertebrae and collect ascites fluid; centrifuge the ascites fluid at 2000 rpm for 5 min, take the light yellow ascites fluid in the middle, aliquot it and store it at -80℃.
[0100] Antibody purification: Take 250 μL of rProtein G Agarose into a 15 mL centrifuge tube, wash three times with PBS, then add 450 μL of aliquoted ascites fluid and dilute with PBS to 8 mL, incubate overnight at 4 °C; wash the beads 6 times with PBS, then 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 neutralize the pH with 1 / 10 volume of 1 M Tris-HCl (pH = 8.5) before aliquoting and storing at -80 °C.
[0101] Example 3:
[0102] This embodiment provides an application of the monoclonal antibody described in Embodiment 2 in the study of adaptive immune responses in bony fish, specifically including the following:
[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 tarda, a fish-killing bacterium, in TYB medium containing Col resistance for 8 hours, centrifuge and wash, then adjust the OD value using a microplate reader. 600 =1, then diluted 40 times with PBS;
[0105] 2. Take several healthy Nile tilapia and divide them into an experimental group and a control group. In the experimental group, each tilapia was injected intraperitoneally with 200 μL of diluted Edwardsiella tarda, while the control group was injected with the same dose of PBS.
[0106] 3. On day 5 post-infection, Nile tilapia in both the experimental and control groups were sacrificed, and their spleens were removed. Leukocytes were separated using density gradient centrifugation (i.e., the method described in section 1 of step six above), resuspended in FACS Buffer, and added to a 96-well V-plate. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cell pellet was resuspended with 100 μL of the anti-tilapia CD122 protein monoclonal antibody from Example 2. The pellet was incubated on ice for 30 min, centrifuged, and washed twice with 200 μL of FACS Buffer. The centrifuged cells were then resuspended with 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000 times, incubated on ice in the dark for 30 min, centrifuged, and washed twice with 200 μL of FACS Buffer. The cells were then resuspended with 200 μL of FACS Buffer, filtered through a 200-mesh sieve, and analyzed by flow cytometry.
[0107] like Figure 7 As shown, flow cytometry analysis revealed that 5 days after infection with *Edwards ichthyophthirius multifiliis*, CD122... + The proportion of cells in T cells increased from 10.8% before infection to 30.2% after infection (as shown in the "E. piscicida day5" section), indicating that tilapia expressed CD122 after infection. + The proportion of T cells increases dramatically in response to pathogen infection and plays a role in the antimicrobial immune process.
[0108] II. Application of anti-tilapia CD122 protein monoclonal antibody in detecting the distribution of CD122 protein in tilapia immune-related tissues, specifically including the following steps:
[0109] 1. Take healthy Nile tilapia and collect peripheral blood, head kidney, liver, and spleen, and separate leukocytes from each tissue. When separating peripheral blood leukocytes, first use a syringe to draw 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 tail vein, mix well, centrifuge at 2500 rpm for 3 min, discard the supernatant, resuspend the cell pellet in L15 medium, and separate leukocytes using the above Percoll separation solution;
[0110] 2. Resuspend leukocytes from each tissue in 1 mL of FACS buffer, add to a 96-well V-plate, centrifuge at 2500 rpm for 3 min, and discard the supernatant. Resuspend leukocytes from each tissue in 100 μL of the anti-tilapia CD122 monoclonal antibody from Example 2, incubate on ice for 30 min, centrifuge the leukocytes from each tissue, and wash twice with 200 μL of FACS buffer. Resuspend the centrifuged cells in 100 μL of anti-mouse Alexa Fluor 647 fluorescent secondary antibody diluted 2000 times, incubate on ice in the dark for 30 min, centrifuge the cells, and wash twice with 200 μL of FACS buffer. After resuspending the cells in 200 μL of FACS buffer, perform flow cytometry detection.
[0111] like Figure 8 As shown, the percentages of CD122+ lymphocytes in peripheral blood (PBL), head kidney (HK), liver (Liver), and spleen (SP) lymphocytes were 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 anti-tilapia CD122 protein monoclonal antibody in specifically recognizing tilapia CD122 protein and enhancing tilapia CD122 protein expression, which includes the following steps:
[0113] 1. Spleens of healthy, unimmunized Nile tilapia were collected, and splenic lymphocytes were obtained using density gradient centrifugation. Splenic lymphocytes were stimulated in vitro with PHA for 6 hours and 12 hours, respectively, and simultaneously stimulated with anti-tilapia CD3ε monoclonal antibody and anti-tilapia CD28 monoclonal antibody for 6 hours and 12 hours, respectively. Meanwhile, Nile tilapia infected with Edwardsiella tarda for 3 and 5 days were prepared, and their spleens were removed, ground, and centrifuged to obtain splenic lymphocytes for 3 days and 5 days of infection, respectively.
[0114] 2. Prepare NP40 lysis buffer. Lyse the six types of splenic lymphocytes with NP40 lysis buffer on ice for 30 min. After lysis, centrifuge at 12000 rpm and 4℃ for 10 min. Take the supernatant and put it into a 1.5 mL EP tube, mix it with 5× loading buffer, and boil it in a boiling water bath for 10 min to obtain protein samples corresponding to the six types of splenic lymphocytes.
[0115] 3. Add each protein sample to the well of an SDS-PAGE gel and perform gel electrophoresis.
[0116] 4. Remove the gel from the gel plate, remove the stacking gel, press the separating gel firmly against the NC membrane, remove air bubbles, set the voltage to 100V, place the transfer instrument in an ice-water bath, and use the transfer instrument to transfer all the protein on the gel to the NC membrane.
[0117] After the transfer was completed, 15 mL of skim milk powder was prepared with PBST (PBS + 0.5% Tween 20), the NC membrane was placed in the skim milk powder, and sealed on a shaker at room temperature for one hour. Then it was washed three times with PBST for 10 minutes each time.
[0118] 4. Place the washed membrane in the culture supernatant of hybridoma cell line 2D8A7 (containing antibodies secreted by hybridoma cell line 2D8A7) and incubate overnight on a shaker at 4°C, ensuring that the membrane is completely covered by the supernatant during incubation.
[0119] After incubation, the membrane was washed three times with PBST for 10 minutes each time. Afterward, the membrane was transferred to a skim milk solution containing anti-mouse Alexa Fluor 680 (Abcam) and incubated for one hour. After incubation, it was washed three times with PBST for 10 minutes each time. The NC membrane was scanned using an Odyssey CLX imaging system.
[0120] like Figure 9 As shown, the anti-tilapia CD122 protein monoclonal antibody can specifically bind to the Nile tilapia CD122 protein, which is approximately 70 kDa in size, consistent with the predicted protein size. Furthermore, it was confirmed that PHA, anti-tilapia CD3ε monoclonal antibody + anti-tilapia CD28 monoclonal antibody (i.e.,...) were used to bind to the CD122 protein in Nile tilapia. Figure 9 After 6 and 12 hours of in vitro stimulation with “αCD3ε / 28”, the expression level of CD122 protein was upregulated. Furthermore, after infection with Edwardsiella tarda, the expression level of CD122 was also upregulated, indicating that the obtained CD122 antibody has the ability to specifically bind to the protein, and that the expression of CD122 protein increases after T cell activation.
[0121] Example 4:
[0122] This embodiment provides the application of the anti-tilapia CD122 monoclonal antibody in Example 2 in the preparation of fish disease prevention and treatment reagents / drugs.
[0123] In summary, this invention employs a lentiviral infection method to transform a plasmid containing the Nile tilapia CD122 gene fragment into a mouse homologous NIH / 3T3 cell line, enabling the expression of active tilapia CD122 protein on the cell surface. Furthermore, a screening and identification mechanism combining flow cytometry, semi-quantitative detection, and immunofluorescence techniques was constructed, successfully producing a monoclonal antibody against tilapia CD122. The experimental results provide strong evidence for understanding CD122 as a marker molecule of early vertebrate T cell activation, and offer important tools and technical support for research on fish adaptive immunity, particularly fish T cell immunity. They also provide a reference for fish disease control and vaccine efficacy evaluation.
[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A hybridoma cell line that secretes a monoclonal antibody against tilapia CD122 protein, characterized in that, The hybridoma cell line described is hybridoma cell line 2D8A7, which was deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with accession number CCTCC NO: C2024142, and deposit address: Wuhan University, Wuhan, China.
2. A monoclonal antibody against tilapia CD122 protein secreted by the hybridoma cell line 2D8A7 of claim 1.
3. A method for preparing the monoclonal antibody according to claim 2, characterized in that, Includes the following steps: Ascites preparation: Mice were taken and injected intraperitoneally with sterile paraffin oil; The hybridoma cell line 2D8A7 described in claim 1 was resuspended and then injected intraperitoneally into mice. The mice were euthanized, ascites fluid was collected, centrifuged, and aliquoted for storage. The ascites fluid was purified to obtain the monoclonal antibody against the tilapia CD122 protein.