Hybridoma cell line secreting monoclonal antibody against fish granzyme b and its use

Abstract

This invention discloses a hybridoma cell line secreting a monoclonal antibody against fish granzyme B and its applications, relating to the fields of fish immunology and disease control. The hybridoma cell line 2C8H3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2024141, dated May 9, 2024. This invention purified tilapia granzyme B protein using a eukaryotic expression system and used it as an antigen to immunize mice. Based on this, a screening and identification method combining flow cytometry, semi-quantitative detection, and enzyme-linked immunosorbent assay (ELISA) was constructed, successfully preparing a monoclonal antibody that specifically recognizes the Nile tilapia granzyme B molecule. This provides an effective tool and technical support for research on fish adaptive immunity mechanisms, particularly cytotoxic T lymphocytes.

Description

Technical Field

[0001] This invention belongs to the field of fish immunology and disease control, and relates to a hybridoma cell line that secretes anti-fish granzyme B monoclonal antibody and its preparation method, the monoclonal antibody, and the application of the monoclonal antibody in fish adaptive immunization. Background Technology

[0002] T cells are an important component of adaptive immunity, playing crucial roles such as killing target cells and secreting cytokines. Based on the different marker molecules on their surface, T cells can be classified into CD4+ cells and CD5+ cells. + T cells and CD8 + Two subsets of T cells, including activated CD8 + T cells, also known as cytotoxic T cells (CTLs), secrete cytotoxic factors and lyse infected cells, playing a crucial role in combating intracellular pathogens such as viruses. As cytotoxic factors, the perforin-granzyme pathway is an important route for CTLs to exert their killing function. Granzyme B is the most potent pro-apoptotic granzyme, similar to cysteine ​​proteases, which cleaves the aspartic acid residue of the substrate. In the former pathway, granzyme B enters the target cell via a transmembrane channel formed by perforin A perforating the cell surface, activating cysteine ​​aspartic proteases (such as caspase-3 and caspase-7) and inducing apoptosis in the target cell. In the latter pathway, granzyme B indirectly initiates this pathway by hydrolyzing Bid protein in the BH3 subfamily of the pro-apoptotic family.

[0003] Compared to mammals, research on adaptive cellular immunity in fish is relatively fragmented. Fish occupy a unique position in evolutionary history, being among the earliest vertebrates to possess adaptive immunity. Various T cell subsets and granzyme B proteins are also present in fish. However, current research on CD8+ in bony fish remains incomplete. + There is limited research on the T cell immune process and how granzyme B exerts its cytotoxic effect in CTLs. This may be because the process of purifying recombinant granzyme B protein from bony fish and preparing monoclonal antibodies is quite difficult, which to some extent limits the development of fish T cell research.

[0004] T lymphocytes play an irreplaceable role in adaptive immunity; however, due to the lack of monoclonal antibodies against T lymphocyte-associated cytotoxic cytokines, in-depth research cannot be conducted on issues such as the toxic function and mechanism of action of T lymphocytes.

[0005] Therefore, developing a specific monoclonal antibody 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 the control of fish diseases. Summary of the Invention

[0006] This invention addresses existing technical challenges in fish cytotoxic T-cell immunity research by developing a preparation and screening technique for monoclonal antibodies (MAbs) based on a eukaryotic expression system. A hybridoma cell line, 2C8H3, capable of stably secreting granzyme B MAbs against Nile tilapia (Oreochromis niloticus), has been successfully prepared. The secreted antibody specifically recognizes and binds to tilapia lymphocytes expressing granzyme B molecules, providing an effective tool and technical support for exploring fish adaptive immune mechanisms, particularly cytotoxic T lymphocytes.

[0007] The technical solution provided by this invention to solve the above-mentioned technical problems is as follows:

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

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

[0010] Construct a plasmid that can express the fish granzyme B gene;

[0011] The plasmid was used to infect cells, and recombinant fish granzyme B protein was isolated and purified from the infected cells;

[0012] Animal immunization was performed using recombinant granzyme B protein from fish as an immunogen;

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

[0014] In a preferred embodiment of the invention, the infected cells comprise 293T cells.

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

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

[0017] Add Protein G garose to the supernatant, incubate, centrifuge, wash Protein G, and wash away unbound protein.

[0018] Granzyme B eukaryotic recombinant protein was eluted with acidic Glycine-HCl solution and then neutralized with alkaline Tris-HCl solution;

[0019] The eluted target protein was placed in pre-cooled PBS for thorough dialyzing. The supernatant was collected and centrifuged to an appropriate volume to obtain the purified recombinant fish granzyme B protein.

[0020] Thirdly, 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] Fourthly, this invention discloses a method for preparing a monoclonal antibody as described in the third aspect, comprising the following steps:

[0022] Purified recombinant protein of fish granzyme B was used as an antigen to immunize mice until the mice produced antibodies.

[0023] The spleen leukocytes of immunized mice were fused with SP2 cells to screen for hybridoma cells that could stably secrete antibodies, and the cell pores that showed positive results were subcloned.

[0024] Monoclonal antibodies are antibodies selected from those produced by hybridoma cells that can specifically bind to the granzyme B protein secreted by fish T lymphocytes.

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

[0026] In a sixth aspect, the present invention discloses the application of a 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 a monoclonal antibody as described in the third aspect in the specific recognition of fish Granzyme B protein.

[0028] Eighthly, the present invention discloses the application of a 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 control reagents / drugs include fish disease prevention and control vaccines.

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

[0031] This invention purifies tilapia granzyme B protein using a eukaryotic expression system and uses it as an antigen to immunize mice. Based on this, a screening and identification method combining flow cytometry, semi-quantitative detection, and enzyme-linked immunosorbent assay (ELISA) was constructed, and a monoclonal antibody that can specifically recognize Nile tilapia granzyme B molecules was successfully prepared. Attached Figure Description

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

[0033] Figure 2 Flow cytometry analysis results of a specific cell population 4 hours after P+I stimulation of 2C8 fusion cell supernatant bound to lymphocytes;

[0034] Figure 3 ELISA analysis results of granzyme B protein binding in the culture supernatant of 2C8 fusion cells;

[0035] Figure 4 The results of flow cytometry analysis of a specific cell population were obtained 4 hours after subcloning of 2C8H3 fusion cell supernatant combined with lymphocytes and stimulated with P+I.

[0036] Figure 5 Electrophoresis diagrams of genes in granzyme B positive and negative cells;

[0037] Figure 6 To detect the effects of anti-granzyme B monoclonal antibody stimulation on tilapia CD3 / CD28 monoclonal antibody and the change in the proportion of granzyme B-positive cells in the Streptococcus agalactiae infection group and the control group;

[0038] Figure 7 The expression of granzyme B at the protein level was compared between the anti-granzyme B monoclonal antibody-specific binding protein sample and the protein expression of granzyme B after 12 hours of PHA stimulation.

[0039] Figure 8 To detect the proportion of granzyme B-positive cell populations in lymphocytes from tissues such as head kidney, peripheral blood, and spleen using anti-granzyme B monoclonal antibody. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It is emphasized that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of the invention.

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

[0042] Example 1

[0043] This embodiment provides a hybridoma cell line 2C8H3 that secretes a monoclonal antibody against fish granzyme B. This hybridoma cell line 2C8H3 was deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2024, with accession number CCTCCNO: C2024141. The fish in this case 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 that can express the fish granzyme B gene;

[0046] The main methods used are as follows:

[0047] Nile tilapia granzyme B gene synthesis:

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

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

[0050] The main methods used are as follows:

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

[0052] ② 6.6 × 10 6 293T cells were evenly seeded in a 10cm cell culture dish and cultured overnight. When the cell confluence reached 80%-90%, the culture medium was replaced with 9mL of DMEM containing 25mM chloroquine (1% penicillin and 10% FBS).

[0053] ② Transfection: Add 30 μg Granzyme B-pINFUSE-mIgG2b-Fc2 plasmid and 150 μL 2.5M CaCl2 to 1500 μL ddH2O, gently pipette, and simultaneously slowly add 1500 μL 2×HEPES-Buffer to prepare the transfection system. After mixing well, carefully add the mixture to the above 293T cells and incubate at 37℃.

[0054] ③ Protein purification: 48 h after transfection, collect the culture supernatant of 293T cells, centrifuge at 1200 rpm for 10 min to obtain supernatant containing Granzyme B eukaryotic recombinant protein; add 1 mL of Protein G Garose to the above supernatant and incubate overnight at 4 °C on a shaker; centrifuge at 1500 rpm for 1 min, and wash Protein G with 10 mL of PBS, repeating 6 times to thoroughly wash away unbound protein; elute Granzyme B eukaryotic recombinant protein with 10 mL of 0.1 M Glycine-HCl (pH 2.8), and neutralize with 1 mL of 1 mM Tris-HCl (pH 8.5); place the eluted target protein in pre-cooled PBS at 4 °C and dialyze thoroughly for 24 h; collect the supernatant, transfer it to 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 kit. The tested proteins were aliquoted and frozen at -80°C.

[0055] ④ After diluting 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] like Figure 1 As shown, the recombinant protein of tilapia granzyme B was purified, and its size was between 35-40 kDa.

[0057] S3: Animal immunization was performed using recombinant granzyme B protein from fish as an immunogen;

[0058] The main methods used are as follows:

[0059] Animal immunization:

[0060] The purified protein was used as an antigen to immunize 6-week-old BALB / c mice, with a total of 5 immunizations. The second immunization was performed 2 weeks after the first immunization, and thereafter every week. For the first and second immunizations, 100 μg of purified protein was thoroughly emulsified with complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, and injected intraperitoneally into the mice. For the subsequent three immunizations, 100 μg of protein was injected into the tail vein of the mice.

[0061] S4: After animal immunization, cell fusion, cell screening, and cloning are performed to obtain the hybridoma cell line 2C8H3.

[0062] The main methods used are as follows:

[0063] Cell fusion:

[0064] ① One week before fusion, SP2 cells were resuscitated and passaged, and cell growth was checked daily and passaged as needed. Two days before fusion, the cells were transferred to T75 cell culture flasks, and the medium was changed 12 hours before fusion.

[0065] ② On the 5th day after the last immunization, cell fusion was performed. First, the thymus of a healthy 4-week-old mouse that had not been immunized was taken, washed with RPMI-1640 medium, and ground on a 200-mesh sterile sieve. The cells were then collected with RPMI-1640 and placed in a large centrifuge tube. After centrifugation, the thymocytes were resuspended in RPMI-1640 containing HAT. The immunized mouse was then sacrificed, and its spleen was removed. The spleen was ground and filtered according to the above method. The spleen cells were resuspended in RPMI-1640, and large precipitates were discarded to obtain the appropriate cell suspension. The thymocytes were temporarily stored in a 37°C incubator.

[0066] ③ Mix the above spleen cell suspension with SP2 cells, then place it 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 your wrist to loosen the cell pellet 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) to the loosened mixed cell pellet within 90 seconds, let stand for 90 seconds, and then continue to slowly add 10 mL of RPMI-1640 over 5 minutes. After the addition is complete, centrifuge at 800 rpm for 6 minutes and discard the supernatant.

[0068] ⑤ Add 3 mL of RPMI-1640 to resuspend the cell pellet, and add it to the thymocyte suspension in ② above. Add the mixed cell suspension evenly to 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 culturing.

[0070] ⑦ After about 2-3 days, the supernatant can be screened again and used for subsequent experiments;

[0071] Screening for monoclonal antibody-positive fusion cells:

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

[0073] ② After centrifugation, a ring of white blood cells appears at the separation point. Slowly aspirate the suspended white blood cells from the separated layer, wash with L15 medium, centrifuge for 3 min, discard the supernatant, and then resuspend in DMEM medium (DMEM medium + 10% FBS + 1% antibiotics). Incubate at 28℃ for 30 min, then add 1 μL LP + I (PMA + ionomycin, V:V = 1:1) to stimulate the cells. After 4 h, aspirate the cells, centrifuge, wash, resuspend in PBS, and add to a 96-well V-plate.

[0074] ③ After centrifuging at 2500 rpm for 3 min, discard the supernatant, fix with BD fixative on ice for 30 min, then suspend the cells with 100 μL of fusion cell supernatant, add 100 μL of SP2 cell supernatant to the negative wells, and incubate on ice for 30 min. After centrifuging the cells, wash twice with 200 μL of washing buffer.

[0075] ④ Resuspend the centrifuged cells with 100 μL of anti-mouseAlexa Fluor 647 secondary antibody diluted 2000 times, incubate on ice for 30 min in the dark, centrifuge the cells and wash them twice with 200 μL washing buffer.

[0076] ⑤ After washing, the cells were suspended in 200 μL washing buffer, filtered, and then analyzed by flow cytometry.

[0077] like Figure 2 As shown, flow cytometry analysis revealed that antibodies secreted by hybridoma cells (fusion cells of spleen leukocytes and SP2 cells) in well 2C8 could bind to proteins in a certain cell group in Nile tilapia leukocytes. This cell group accounted for approximately 7.22% of Nile tilapia spleen lymphocytes 4 hours after P+I stimulation.

[0078] The binding of antibodies secreted by 2C8 cells to tilapia granzyme B protein was determined by ELISA.

[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 coating solution containing recombinant protein granzyme B to each well to coat the microplate, and block overnight at 4°C;

[0080] ② After incubation, wash the microplate three times with PBST (PBS + 0.5‰ Tween 20), 5 min each time. Then add 200 μL of 1% BSA to each well and incubate at 37℃ for 1 h.

[0081] ③ After incubation, wash the microplate with PBST 3 times for 5 min each time, then add 100 μL of primary antibody (i.e. fusion cell supernatant) to each well and incubate at 37℃ for 1 h;

[0082] ④ After incubation, wash the microplate three times with PBST for 5 minutes each time. Then add 100 μL of alkaline phosphatase-labeled (AP) goat anti-mouse IgG (1:3000) to each well and incubate at 37°C for 1 hour.

[0083] ⑤ After incubation, wash the microplate three times with PBST for 5 minutes 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 MgCl2 to each well. Incubate in the dark for 30 minutes. After incubation, add 50 μL of 2 M NaOH to each well to stop the incubation. Detect the OD using a microplate reader. 405 The absorbance value at that location.

[0084] like Figure 3 As shown, the expression level of granzyme B gene was high in the positive well 2C8, while the expression of granzyme B was almost non-existent in the negative well, proving that the monoclonal antibody can specifically bind to the granzyme B protein secreted by T lymphocytes of Nile tilapia.

[0085] Subclonal positive hybridoma cells:

[0086] ① Take the thymus of an untreated four-week-old healthy mouse and treat it in the same way as above. First, wash it with RPMI-1640 medium, grind it aseptically, centrifuge it, and then resuspend it with medium to obtain a thymus cell suspension. Centrifuge it at 1000 rpm for 5 min, resuspend the precipitate with 10 mL of RPMI-1640 and mix it well.

[0087] ② The hybridoma cells (fusion cells of spleen leukocytes and SP2 cells) in the above 2C8 wells were blown up and serially diluted with RPMI-1640 medium. After dilution, they were counted using a hemocytometer.

[0088] ③ After counting, take about 100 hybridoma cells and add them to the above thymocyte suspension. Mix well and slowly drop them into a 96-well cell culture plate, with a volume of about 100 μL per well.

[0089] ④ Place the culture plate in a 37°C incubator. After about 10-14 days, aspirate the hybridoma supernatant and perform flow cytometry analysis as described above.

[0090] like Figure 4 As shown, after cloning the cells in the 2C8 well, the resulting hybridoma cell line can stably secrete antibodies. These antibodies can still bind to proteins in a certain cell group in Nile tilapia leukocytes in a stable ratio. This hybridoma cell line is named 2C8H3.

[0091] The antibody secreted by 2C8H3 cells was identified as a monoclonal antibody against tilapia granzyme B using a semi-quantitative method.

[0092] ① Take the spleen of Nile tilapia according to the above method, wash, grind and filter it, add it to Percoll separation solution, centrifuge it and aspirate the suspended white blood cell layer in the separation layer, wash it with L15 medium, centrifuge it, resuspend it in L15 medium, and then transfer it to a 2mL EP tube.

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

[0094] ③ Resuspend the centrifuged cells with 1.5 mL of anti-mouseAlexa Fluor 647 fluorescent secondary antibody diluted 2000 times, incubate on ice for 30 min in the dark, centrifuge the cells and wash them twice with 1 mL washing buffer;

[0095] ④ After suspending the cells in 1 mL of washing buffer, sort them and collect positive and negative cell populations separately. Collect at least 2 × 10⁶ positive cells. 6 indivual;

[0096] ⑤ Total RNA was extracted from positive and negative cells using the Trizol method, and cDNA templates were obtained by reverse transcription. Gene fragments of β-actin, granzyme B, CD3, and IgM from Nile tilapia were amplified by PCR reaction. Primer sequences are shown in the table below.

[0097] Primer information

[0098]

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

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

[0101] In this embodiment, granzyme B protein was purified and used as an antigen to immunize mice. After the mice produced antibodies, spleen cells from the immunized mice were fused with SP2 cells, and hybridoma cells capable of stably secreting antibodies were screened by flow cytometry. Subcloning was performed on the wells of cells that showed positive results on flow cytometry. ELISA and semi-quantitative screening methods confirmed that the antibodies produced by the 2C8H3 cell line could specifically bind to the granzyme B protein secreted by Nile tilapia T lymphocytes, and could specifically recognize Nile tilapia granzyme B. + cell.

[0102] Example 2

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

[0104] Granzyme B in T cells of tilapia spleen lymphocytes stimulated with anti-tilapia CD3 / 28 monoclonal antibody was detected using anti-tilapia granzyme B monoclonal antibody. + The lymphocyte count is determined through the following steps:

[0105] The applicant has previously prepared monoclonal antibodies against tilapia CD3 and CD28. For example, Chinese patent publication number CN112501130A discloses the preparation of a mouse anti-tilapia CD3ε monoclonal antibody, and Chinese patent publication number CN116355861A discloses the preparation of a mouse anti-tilapia CD28 monoclonal antibody. These antibodies are used in vitro to stimulate lymphocytes, mimicking the dual signals of T cell activation. A healthy tilapia is taken, euthanized, and its spleen is harvested, ground, filtered, and subjected to density gradient centrifugation as described above to obtain spleen lymphocytes. The lymphocytes are resuspended in 1 mL of DMEM medium. 500 μL is used as a control, and 2 μg of CD3 / 28 monoclonal antibody is added to the remaining 500 μL of lymphocytes. Simultaneously, 1 μL of Golgi plug is added to each well, and the volume is brought to 1 mL with culture medium. The cells are then incubated at 28°C. After 12 hours of stimulation, lymphocytes were collected in 96-well V-plates and centrifuged at 2500 rpm for 3 minutes. CD3 antibody, diluted 1:400, was added to each well (100 μL per well) and the cells were stained for 30 minutes. After centrifugation and washing, the centrifuged cells were resuspended in 100 μL of 2000-fold diluted anti-mouse Alexa Fluor 647 fluorescent secondary antibody and incubated on ice for 30 minutes in the dark. After staining, the cells were centrifuged, fixed on ice for 30 min with BD fixative, centrifuged again, washed twice with washing buffer, and then resuspended with 100 μL of the anti-tilapia granzyme B-FITC monoclonal antibody labeled in Example 2. The cells were incubated on ice for 30 min, centrifuged, and washed twice with 200 μL washing buffer. After centrifugation, the cells were washed twice with 200 μL washing buffer. After resuspending the cells with 200 μL washing buffer, the cells were filtered through a 200-mesh sieve and then analyzed by flow cytometry.

[0106] like Figure 6 As shown above, flow cytometry analysis revealed that granzyme B... + The proportion of granzyme B-expressing 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 T cell activation.

[0107] Example 3

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

[0109] Detection of granzyme B in tilapia infected with Streptococcus agalactiae using anti-tilapia granzyme B monoclonal antibody + The lymphocyte count is determined through the following steps:

[0110] Add the bacterial culture to 5 mL of BHI medium at a ratio of 1:1000. After the culture becomes turbid, adjust the bacterial concentration using an ELISA plate. The bacterial concentration should be adjusted to OD0.05. 600 When the concentration was 1, the bacterial suspension was diluted 40-fold. 200 μL of diluted *Streptococcus agalactiae* was injected intraperitoneally into each healthy Nile tilapia, while the control group received the same dose of PBS. On day 5 after infection, spleen tissue was collected from tilapia in each treatment group. One day before sacrifice, 100 μL of BFA was injected into each treatment group. Leukocytes were obtained using the Percoll centrifugation method described above and resuspended in FACS Buffer. The cell suspension was added to 96-well V-plates, centrifuged at 2500 rpm for 3 min, and the supernatant was discarded. CD3 antibody was diluted 1:400 and added to each well (100 μL). Cells were aspirated and stained for 30 min. The centrifuged cells were then resuspended with 100 μL of 2000-fold diluted anti-mouse Alexa Fluor 647 secondary antibody and incubated on ice for 30 min in the dark. After staining, the cells were centrifuged, fixed on ice for 30 min with BD fixative, washed twice with FASC buffer, centrifuged again, washed twice with washing buffer, and then resuspended with 100 μL of the anti-tilapia granzyme B-FITC monoclonal antibody labeled in Example 2. The cells were incubated on ice for 30 min, centrifuged, and washed twice with 200 μL washing buffer. After centrifugation, the cells were washed twice with 200 μL washing buffer. After resuspending the cells with 200 μL washing buffer, the cells were filtered through a 200-mesh sieve and then analyzed by flow cytometry.

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

[0112] Example 4

[0113] This embodiment provides the application of the anti-fish granzyme B monoclonal antibody secreted by the hybridoma cell line 2C8H3 in Example 1 in the specific recognition of fish granzyme B protein.

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

[0115] Leukocytes were isolated from the spleens of healthy Nile tilapia and resuspended in 1 mL LDM medium. The cells were divided into two wells. One well was incubated with 500 μL of medium, and the other well was stimulated with 500 μL of medium and 2 μL PHA for 12 h. After stimulation, the cells were resuspended, centrifuged at 8000 rpm for 3 min, and the supernatant was discarded. 80 μL of NP-40 lysis buffer (1% protease inhibitor, 1‰ PMSF, 1% phosphorylase inhibitor) was added to each well to resuspend the cells, and the cells were lysed on ice for 30 min. The cells were then centrifuged at 12000 rpm at 4 °C for 10 min, and the supernatant was collected. 20 μL of 5×SDS loading buffer was added, and the mixture was boiled in a boiling water bath for 10 min to obtain two sets 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 the protein sample.

[0117] Protein and cell samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 10 μL of sample was added to each well. First, a constant current of 40 mA was maintained. When the sample reached the boundary between the stacking gel and the separating gel, the current was adjusted to 80 mA. The gel was stopped at the appropriate position with reference to the marker position.

[0118] Open the gel plate, remove the gel and discard the stacking gel. Then, use a transfer apparatus to attach the gel to the NC membrane and place it in transfer buffer (transfer buffer: 10% transfer buffer, 70% ultrapure water, 20% methanol). Run at a constant voltage of 100V for 2 hours. During the transfer process, the transfer apparatus should be placed on an ice-water bath. After all the samples have been transferred to the NC membrane, place the NC membrane in 15 mL of PBST solution containing 4% skim milk powder and block at room temperature for 1 hour. Then wash three times with PBST for 10 minutes each time. Finally, cut the membrane according to the size of the target protein and place them into the antibody supernatant secreted by granzyme B fusion cells. Incubate overnight at 4°C.

[0119] After incubation, wash the membrane three times with PBST for 10 minutes each time;

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

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

[0122] Example 5

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

[0124] granzyme B + The distribution of lymphocytes in different tissues of Nile tilapia was detected using the following experimental steps:

[0125] Take a healthy Nile tilapia and collect peripheral blood, liver, gills, head kidney and spleen respectively, and separate leukocytes according to the method in Example 1.

[0126] When isolating peripheral blood leukocytes, first use a 5mL syringe to take 2mL of anticoagulant (15mM sodium citrate, 450mM NaCl, 0.1M glucose, 10mM EDTA, pH 7.0), remove air bubbles, then use a syringe to draw peripheral blood from the tail vein and mix thoroughly. Centrifuge at 2500rpm for 3min, discard the supernatant, and resuspend the cell pellet in L15 medium. Use the Percoll density gradient separation method described above to separate leukocytes.

[0127] Leukocytes from each tissue were resuspended in 1 mL of LACS buffer. 100 μL of cell suspension was added to each well of a 96-well V-plate, centrifuged at 2500 rpm for 3 min, and the supernatant was discarded. The cells were fixed with BD fixative on ice for 30 min, centrifuged, washed twice with washing buffer, and then resuspended in 100 μL of supernatant from the anti-tilapia granzyme B fusion cells in Example 2. 100 μL of SP2 cell supernatant was added to the wells of the negative control group, and the cells were incubated on ice for 30 min. After centrifugation, the cells were washed twice with 200 μL of washing buffer. The centrifuged cells were resuspended in 100 μL of 2000-fold diluted anti-mouse Alexa Fluor 647 fluorescent secondary antibody, incubated on ice in the dark for 30 min, centrifuged, and washed twice with 200 μL of washing buffer. After resuspending the cells in 200 μL of washing buffer, the cells were filtered and analyzed by flow cytometry.

[0128] like Figure 8 As shown, granzyme B was found in lymphocytes from peripheral blood, liver, gills, head kidney, and spleen. + The percentages of 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 in multiple tissues. + Cell population.

[0129] In summary, this invention establishes a complete eukaryotic expression system and purification system. Based on this system, this project prepared and purified recombinant eukaryotic Granzyme B protein from fish to immunize mice, and constructed a screening and identification mechanism combining flow cytometry, semi-quantitative detection, and ELISA detection. Monoclonal antibodies against fish Granzyme B were successfully prepared, providing important tools and technical support for research on fish adaptive immunity, especially fish cytotoxic T cells. It also provides technical support and theoretical basis for fish disease control and vaccine efficacy evaluation.

[0130] 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 2C8H3 that secretes a monoclonal antibody against fish granzyme B, characterized in that, The hybridoma cell line 2C8H3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2024141 and deposit date of May 9, 2024.

2. A monoclonal antibody against fish granzyme B secreted by the hybridoma cell line 2C8H3 as described in claim 1.

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