Anti-tilapia igm monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The application discloses an anti-Rofer IgM monoclonal antibody, a hybridoma cell strain and application thereof. The CDR sequences of a heavy chain variable region of the monoclonal antibody are shown as SEQ ID NO. 1-3, and the CDR sequences of a light chain variable region are shown as SEQ ID NO. 4-6. The monoclonal antibody can be obtained by secretion of the hybridoma cell strain with a preservation number of CCTCC NO: C2023153. The monoclonal antibody provided by the application can be used for specific detection of secreted IgM of the Rofer by Western blot and ELISA, and can be used for identification of IgM of the Rofer by flow cytometry + The B cell has the characteristics of good specificity, high sensitivity and strong affinity, and can be used for structural analysis of the IgM of the Rofer and detection of an immune response level, thereby laying a foundation for in-depth research on the immune system of the Rofer and establishment of an evaluation method for an immune effect of a vaccine of the Rofer with an antibody level as an index.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monoclonal antibodies, and particularly relates to an anti-tilapia IgM monoclonal antibody, a hybridoma cell strain and application thereof. BACKGROUND

[0002] As a world economic fish, tilapia (Oreochromis niloticus) has become an important variety in global aquaculture due to its rapid growth, excellent meat quality and strong reproductive capacity. However, with the expansion of the scale of aquaculture, disease outbreaks have become a major bottleneck restricting the development of the industry. The death of tilapia caused by Streptococcus agalactiae disease, Luohu virus disease and the like is very serious, causing huge economic losses to the tilapia aquaculture industry.

[0003] At present, the prevention and control of tilapia diseases mainly relies on antibiotics, but the problem of bacterial drug resistance caused by long-term use of antibiotics is increasingly serious, which seriously affects the healthy and sustainable development of the aquaculture industry. Under this background, vaccination as a sustainable disease prevention method can activate the fish immune system and improve the body's resistance to specific pathogens, which will be an important means of preventing tilapia diseases and the mainstream direction of fish disease prevention and control in the future. However, the precise evaluation of the immune effect of the vaccine is often a tedious process, time-consuming, and lacks evaluation standards. Based on the monoclonal antibody of fish immunoglobulin (immunoglobulin, Ig), by analyzing the change rule of the level of specific antibodies (essence is Ig) in fish vaccinated with vaccine to evaluate the immune effect of the vaccine, it will provide a powerful tool for establishing the evaluation standard system of the immune effect of tilapia vaccine.

[0004] As the earliest discovered and most abundant type of immunoglobulin in bony fish, IgM has been proven to play a very important role in the humoral immune function of the fish's systemic and mucosal immunity. Therefore, the development of monoclonal antibodies against tilapia IgM helps to better understand the humoral immune response of tilapia, and helps to establish antibody analysis and detection methods for tilapia, and then to establish a method for evaluating the immune effect of the vaccine based on the level of tilapia antibodies and to develop the use regulations of the vaccine, and to promote the development of tilapia disease immune control technology based on vaccination. SUMMARY

[0005] The application provides a high-affinity mouse anti-tilapia IgM monoclonal antibody, which is secreted by a hybridoma cell with a preservation number of CCTCC NO: C2023153.

[0006] Another object of the present application is to provide a hybridoma cell line OnM-14G10B1E1, which has a preservation number of CCTCC NO: C2023153.

[0007] Another object of the present application is to provide an application of the monoclonal antibody or the hybridoma cell line in detecting IgM of Tilapia, which includes but is not limited to preparing a Tilapia IgM detection kit.

[0008] In order to achieve the above object, the present application adopts the following technical measures:

[0009] The first aspect of the present application provides an anti-Tilapia IgM monoclonal antibody, wherein the sequences of CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO. 1-3, and the sequences of CDRL1, CDRL2 and CDRL3 of the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO. 4-6. Specifically as follows:

[0010] CDRH1: DYSMH (SEQ ID NO. 1);

[0011] CDRH2: WINTETGEPTYADDFKG (SEQ ID NO. 2);

[0012] CDRH3: YGRPRGYFAV (SEQ ID NO. 3);

[0013] CDRL1: KASQDVVTAVA (SEQ ID NO. 4);

[0014] CDRL2: WASTRHT (SEQ ID NO. 5);

[0015] CDRL3: QQYSSYPFT (SEQ ID NO. 6).

[0016] Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

[0017] The amino acid sequence of the heavy chain variable region is as follows:

[0018] QIQLVQSGPELKKPGETVKISCKASDYTFTDYSMHWVKQAPGKGLKWMAWINTETGEPTYADDFKGRFAFSFEISASTAYLQINNLKNEDTATYFCAGYGRPRGYFAVWGAGTTVTVSS (SEQ ID NO. 7)

[0019] The amino acid sequence of the heavy chain variable region is:

[0020] DIVMTQSHKFMSTSVGDRVSITCKASQDVVTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSESGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIR (SEQ ID NO. 8)

[0021] The second aspect of the present application provides a polynucleotide encoding the monoclonal antibody described above.

[0022] Further, the nucleic acid molecule described above includes polynucleotide A (as shown in SEQ ID NO. 9) encoding the heavy chain variable region of the mouse monoclonal antibody against IgM of Tilapia and polynucleotide B (as shown in SEQ ID NO. 10) encoding the light chain variable region of the mouse monoclonal antibody against IgM of Tilapia. Preferably, when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 7, the nucleotide sequence of the nucleic acid molecule A can be:

[0023] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGATTATACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGCCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTTGAAATCTCTGCCAGTACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTGGCTACGGTAGGCCTCGGGGGTATTTCGCTGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO. 9)

[0024] When the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 8, the nucleotide sequence of the nucleic acid molecule B can be:

[0025] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTATCACCTGCAAGGCCAGTCAGGATGTGGTTACTGCTGTAGCCTGGTATCAACAGAAACCAGGTCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGAATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAGATAAGA(SEQ ID NO. 10)

[0026] The third aspect of the present application provides a recombinant vector comprising the above-mentioned polynucleotide.

[0027] The fourth aspect of the present application provides a host cell comprising the above-mentioned polynucleotide or the recombinant vector.

[0028] The fifth aspect of the present application provides a hybridoma cell strain OnM-14G10B1E1, which has been preserved in the China Center for Type Culture Collection located in Wuhan University, Wuhan, China on June 1, 2023, and the preservation number is CCTCC NO: C2023153.

[0029] Further, the hybridoma cell strain OnM-14G10B1E1 or its subculture cell strain can secrete the above-mentioned anti-tilapia IgM monoclonal antibody.

[0030] Further, the above-mentioned anti-tilapia IgM monoclonal antibody can be obtained by injecting the hybridoma cell strain OnM-14G10B1E1 into a mouse, taking the mouse ascites, and then isolating and purifying the mouse ascites.

[0031] The sixth aspect of the present application provides the use of the above-mentioned mouse anti-tilapia IgM monoclonal antibody, polynucleotide, recombinant vector, host cell, or hybridoma cell strain OnM-14G10B1E1 in the preparation of a tilapia IgM detection kit.

[0032] Preferably, the detection kit comprises a colloidal gold immunoreagent kit, a chemiluminescence kit, an enzyme-linked immunosorbent assay kit, or an immunofluorescence kit.

[0033] The seventh aspect of the present application provides a kit for detecting Tilapia IgM, which comprises the above-mentioned mouse anti-Tilapia IgM monoclonal antibody, polynucleotide, recombinant vector, host cell or hybridoma cell strain OnM-14G10B1E1.

[0034] Beneficial effects: The present application uses Protein A affinity purification Tilapia serum natural IgM as an antigen to immunize mice and screen to obtain a hybridoma cell strain OnM-14G10B1E1 which can specifically secrete an anti-Tilapia IgM monoclonal antibody. The hybridoma cell strain has the significant characteristics of stable subculture and stable secretion of the antibody. Moreover, the monoclonal antibody secreted by the hybridoma cell strain OnM-14G10B1E1 can specifically recognize the secreted form of IgM and IgM + B cells, with a titer of 1:2048000, and an affinity constant of up to 6.02x10 -9 M, with the characteristics of good specificity, high titer, sensitive reaction and strong affinity, which helps to analyze the rules of Tilapia's humoral immune response after pathogenic infection, lays a foundation for the development of new vaccines and immune enhancers, provides a reliable tool for Tilapia immunology research, and has important significance for promoting the healthy development of Tilapia aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor under the premise of these drawings.

[0036] Figure 1 The figure is the result of SDS-polyacrylamide gel electrophoresis (PAGE) in Example 1 of the present application under reducing and non-reducing conditions to detect purified Tilapia serum natural IgM and stained with Coomassie brilliant blue.

[0037] Figure 2 The figure is the result of ELISA analysis of the subclass of mouse anti-Tilapia IgM monoclonal antibody in Example 2 of the present application.

[0038] Figure 3 The figure is the result of Western blot under reducing and non-reducing conditions to detect IgM bands after Tilapia serum is diluted 100 and 500 times, respectively, by using mouse anti-Tilapia IgM monoclonal antibody in Example 2 of the present application.

[0039] Figure 4The IgM in the peripheral blood lymphocytes of the tilapia detected by flow cytometry in Example 2 of the present application + The result graph of B cells.

[0040] Figure 5 The detection of tilapia serum by gel filtration chromatography column Superdex TM The result graph of IgM in different elution volumes after separation by 200 Increase 10 / 300 GL (GE Healthcare).

[0041] Figure 6 The binding and dissociation curves of the antibody with the antigen protein at different dilution concentrations when the antibody affinity is determined by surface plasmon resonance technology (SPR) in Example 4 of the present application.

[0042] Figure 7 The recognition result graph of the supernatant of the 0th, 5th, 10th, 15th, 20th and 25th generation cells of the hybridoma cell strain OnM-14G10B1E1 subcultured by flow cytometry in Example 5 of the present application on IgM + The recognition result graph of B cells.

[0043] Figure 8 The recognition result graph of the supernatant of the 0th, 5th, 10th, 15th, 20th and 25th generation cells of the hybridoma cell strain OnM-14G10B1E1 subcultured by Western blot on the secreted form IgM in the serum of tilapia in Example 5 of the present application. DETAILED DESCRIPTION

[0044] The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore are only examples and cannot limit the protection scope of the present application. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be the usual meanings understood by the skilled in the art to which the present application belongs; unless otherwise specified, the reagents, methods and devices used in the present application are the conventional reagents, methods and devices in the art; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] Example 1 Preparation of mouse anti-tilapia IgM monoclonal antibody

[0046] In this embodiment, HiTrap ®Protein A column (GE Healthcare) affinity purification of Tilapia serum natural IgM protein, and its antigen immunized mice after cell fusion technology to obtain hybridoma cell line OnM-14G10B1E1, and the hybridoma cell line OnM-14G10B1E1 secreted mouse anti-Tilapia IgM monoclonal antibody, the specific process is as follows:

[0047] (1) the acquisition of Tilapia serum

[0048] The healthy Tilapia was immersed in 30 mg / L of fish anesthetic MS-222 (Sigma), and after anesthesia, it was placed in a dissection plate, the head was covered with a towel, the scales on the lateral line of the posterior margin of the anal fin were removed, and 10 mL of injection without anticoagulant was inserted into the muscle tissue at the posterior of the anal fin, until the needle reached the spine, and the blood entered the syringe; after the blood was coagulated, it was placed at 4℃ overnight, the next day, it was centrifuged at 4℃, 3000 g for 10 min, and the supernatant was transferred to a clean centrifuge tube.

[0049] (2) purification of Tilapia serum natural IgM

[0050] ① Equilibrium column: wash the Protein A affinity purification column with 10 mL Binding buffer (1 × PBS).

[0051] ② Loading: centrifuge the Tilapia serum at 4℃, 18000 g for 30 min to remove impurities, then pass through a 0.22 μm filter, dilute the serum with Binding buffer at a ratio of 1:3, and then load it onto the Protein A affinity purification column through the ÄKTA pure 25L protein purification system (GE Healthcare).

[0052] ③ Neutral washing: wash the Protein A affinity purification column with 20 mL Binding buffer to remove unbound impurities.

[0053] ④ Acid elution: elute the Protein A affinity purification column with 0.1 M citric acid solution at pH = 3.0, and collect the eluate in a centrifuge tube preloaded with 200 μL of 1 mol / L Tris-HCl solution at pH = 9.0.

[0054] ⑤ Take the collected elution sample to detect the purity of the purified Tilapia IgM by SDS-PAGE, the detection result is shown in Figure 1 Under reducing conditions, it is a heavy chain (about 75 kDa) and a light chain (about 25 kDa), and under non-reducing conditions, it is a monomer or multimer greater than 180 kDa, with a purity of about 95%.

[0055] ⑥The purified tilapia serum IgM was diluted with PBS (pH = 7.4) and ultrafiltrated, and finally the protein concentration was determined by a BCA kit (Biosharp).

[0056] (3) Mouse immunization

[0057] Five 6-8-week-old BALB / c mice were immunized with the purified tilapia serum natural IgM as an immunogen. The immunization route and procedure were as follows: about 50 μg of the tilapia serum natural IgM was mixed with an equal volume of Freund's complete adjuvant to emulsify, and then injected subcutaneously into the back of the mice in multiple points. Two weeks later, the mice were boosted three times at two-week intervals. The antigen protein was mixed with an equal volume of Freund's incomplete adjuvant to emulsify, and then injected subcutaneously into the back of the mice in multiple points. On the 7th day after the third boost, the tail blood was collected for ELISA detection of the antibody titer.

[0058] (4) Cell fusion and culture

[0059] ① Cell fusion: mouse spleen cells and mouse myeloma cells SP2 / 0 were mixed at a cell ratio of 5:1, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 1 mL of PEG-4000 (Sigma) was slowly added to the test tube while shaking; then 9 mL of complete culture medium was slowly added multiple times. The whole process was carried out in a 37°C water bath, followed by centrifugation at 1000 rpm for 10 min to remove the supernatant, and cell fusion was completed.

[0060] ② Cell culture: the cell clumps after centrifugation were gently dispersed, resuspended with HAT medium, and plated in the prepared feeder cell 96-well plate at 200 μL per well. The plate was incubated in a 37°C, 5% CO2 incubator. After one week, the growth of the clones was observed under a microscope and the number of clones per well was recorded. When the cells grew to about 1 / 3 of the area of the culture well, the culture supernatant was removed for ELISA detection.

[0061] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies

[0062] ① Positive clone screening: ELISA plates were coated with natural IgM of tilapia serum (2 μg / mL) at 4°C overnight, washed and blocked the next day, then 100 μL of hybridoma cell culture supernatant to be tested was added to each well, and incubated at 37°C for 1 h; after discarding the supernatant, 100 μL of HRP-labeled goat anti-mouse IgG (Proteintech) diluted 1:5000 was added to each well, and incubated at 37°C for 1 h; after discarding the supernatant, 100 μL of TMB color developing solution (Bi Yun Tian) was added to each well, and color developed at room temperature for 10-30 min; then 50 μL of stop solution (2 M H2SO4) was added to each well to stop the reaction. The absorbance value was detected at 450 nm wavelength by an enzyme-labeled instrument. The positive clone was determined as the one with an absorbance value more than 2 times that of the negative control.

[0063] ② Subcloning of hybridoma cells: the hybridoma cells positive in ELISA were gently washed out from the culture plate, counted, and then inoculated in a 96-well plate at 100 μL per well with continuous dilution of the cell suspension in culture medium. After about 10 days of culture, when the hybridoma cell colonies grew to 1 / 3 of the area of the well bottom, the antibody activity in the supernatant was determined. The positive clones with antibody activity were subcloned for a total of 3 times. After repeated screening, the monoclonal antibody with high antibody titer, high antibody concentration and stable secretion was screened, which could recognize the monomer or multimer form of IgM of tilapia, and could also recognize the B cells of tilapia. + The ELISA and flow cytometry detection results of different monoclonal cell strains are shown in Table 1.

[0064] Table 1. ELISA and flow cytometry detection results of the third subcloned cell strains

[0065]

[0066] Finally, a hybridoma cell strain stably expressing anti-tilapia IgM monoclonal antibody was obtained, which was named hybridoma cell strain OnM-14G10B1E1 (Hybridoma cell line OnM-14G10B1E1). The cell strain was deposited at the China Center for Type Culture Collection on June 1, 2023, located at Wuhan University, Wuhan, China, with the accession number CCTCC NO: C2023153.

[0067] ③ Mass production of monoclonal antibody: 10-week-old healthy BALB / c mice were injected intraperitoneally with liquid paraffin, 0.5 mL per mouse, and 1 week later, each pretreated mouse was injected intraperitoneally with 5 × 10 7The ascites was extracted when the mouse's abdomen was extremely swollen after 10-14 days, and the ascites was extracted once every 2 days, and a total of 6.5 mL of ascites was extracted. The ascites was centrifuged at 17000 g for 10 min at 4 DEG C, and the supernatant was subjected to Protein A affinity chromatography purification, and a total of 11.9 mg of purified product was obtained, that is, the mouse anti-Tilapia IgM monoclonal antibody was obtained, and the concentration of the mouse anti-Tilapia IgM monoclonal antibody in the ascites was 1.83 mg / mL. The concentration of the obtained mouse anti-Tilapia IgM monoclonal antibody was adjusted to 1 mg / mL, and the antibody was diluted by 1000 times first, and then diluted by 2 times gradient, and the titer of the antibody was determined by ELISA. The determination results are shown in Table 2, and the results show that the titer of the mouse anti-Tilapia IgM monoclonal antibody is 1:2048000.

[0068] Table 2 Determination results of the titer of the mouse anti-Tilapia IgM monoclonal antibody

[0069]

[0070] Note: The judgment basis of the antibody titer is that if the OD of the test hole is greater than 0.1, and greater than 2.1 times of the OD of the negative control hole (P / N>2.1, wherein P is the OD value of the test antibody at a certain dilution, and N is the OD value of the negative control), it is determined to be positive, and the highest dilution multiple of the antibody determined to be positive is the titer of the antibody.

[0071] Example 2 Identification of the mouse anti-Tilapia IgM monoclonal antibody

[0072] (1) The process of detecting the antibody subclass is as follows:

[0073] The purified Tilapia serum natural IgM protein was coated in the ELISA plate, and incubated at 4 DEG C overnight; 3 times of washing with PBST was performed, and then 100 μL of the mouse anti-Tilapia IgM monoclonal antibody was added to each hole, and incubated at 37 DEG C for 2 h; after 3 times of washing, 100 μL of the diluted HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA and IgM monoclonal antibody was added to each hole, and incubated at 37 DEG C for 1 h; after 3 times of washing, 100 μL of TMB color developing liquid (Biyun Tian) was added to each hole, and incubated at 37 DEG C for 30 min; then 50 μL of the stop solution (2 M H2SO4) was added to each hole to terminate the reaction. The absorbance value was detected by the enzyme label instrument at 450 nm wavelength, and the HRP-labeled goat anti-mouse Ig subclass used in the positive reaction hole was the antibody class to be detected.

[0074] The identification results are shown in Table 2, and the results show that the mouse anti-Tilapia IgM monoclonal antibody prepared in the application is IgG2b. Figure 2

[0075] ​(2) Western blot detection, the process is as follows:

[0076] ① Preparation of sample: the serum of healthy tilapia was diluted 100 and 500 times with PBS respectively, 1 / 5 volume of 5x reduced and non-reduced SDS-PAGE loading buffer (Solarbio) was added respectively, vortexed, heated at 100°C for 10 min in a metal bath, and placed on ice or stored at -80°C.

[0077] ② SDS-PAGE: 12% and 8% Tris-glycine polyacrylamide lower separation gel and 5% upper concentrated gel were prepared respectively, and electrophoresis was performed, the electrophoresis conditions were 80 V, 0.5 h; 120 V, 1 h.

[0078] ③ Membrane transfer: the SDS-PAGE gel was removed from the gel plate and soaked in the previously prepared membrane transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol), a 0.22 μm PVDF membrane was cut to the appropriate size, soaked in methanol for 30 s to activate, and then transferred to the membrane transfer buffer. According to the order of filter paper-PVDF membrane-gel-filter paper from bottom to top, make sure that there is no bubble in each layer, 300 mA semi-dry transfer for 60 min, transfer the protein on the SDS-PAGE gel to the PVDF membrane.

[0079] ④ Blocking: after membrane transfer, the PVDF membrane was transferred to a TBST (25 mM Tris, 150 mM NaCl, 0.1% Tween-20) solution containing 5% skimmed milk powder (Biosharp), and incubated at room temperature for 2 h.

[0080] ⑤ Primary antibody incubation: discard the blocking solution, dilute the mouse anti-tilapia IgM monoclonal antibody to 2 μg / mL with TBST containing 5% skimmed milk powder, and incubate at 4°C overnight.

[0081] ⑥ Secondary antibody incubation: remove the primary antibody solution, wash the membrane with TBST for 3 times, each for about 10 min, dilute the HRP-labeled goat anti-mouse IgG (H+L) antibody with TBST containing 5% skimmed milk powder at a ratio of 1:5000, and incubate at room temperature for 1 h.

[0082] ⑦ ECL color development: remove the secondary antibody solution, wash the membrane with TBST for 3 times, each for about 10 min, prepare an appropriate amount of ECL color developing solution (Biosharp) by mixing A and B at a ratio of 1:1, add the color developing solution to the PVDF membrane, and analyze and take a photo using a chemiluminescence imager after sufficient contact.

[0083] (3) Flow cytometry detection, the process is as follows:

[0084] ① Isolation of total leukocytes from peripheral blood of tilapia: Anesthetize healthy tilapia and collect peripheral blood from the caudal peduncle using a 5 mL syringe containing heparin sodium (Sigma). Dilute the blood 7 times with DMEM medium (Gibco) and add it to the surface of 34% and 51% discontinuous Percoll (GE Healthcare) liquid. After gradient centrifugation, aspirate the leukocytes between the two discontinuous Percoll layers to obtain the total leukocytes from the peripheral blood of tilapia.

[0085] ② Primary antibody incubation: After washing tilapia peripheral blood total leukocytes twice with PBS containing 2% FBS (Yeasen), mouse anti-tilapia IgM monoclonal antibody was added to a final concentration of 2 μg / mL. The cells were incubated on ice for 45 min, with the cells gently swirled once every 15 min.

[0086] ③ Secondary antibody incubation: After washing the cells twice with PBS containing 2% FBS, add APC Goat Anti-Mouse IgG (Biolegend) to a final concentration of 1 μg / mL, incubate on ice for 30 min, and gently vortex the cells once every 10 min.

[0087] ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspending the cells and filtering them, IgM in tilapia peripheral blood lymphocytes was detected by flow cytometry (BD). + The proportion of B cells.

[0088] Mouse anti-tilapia IgM monoclonal antibody against tilapia secreted IgM and IgM + The identification and specificity verification of B cells are as follows: Figures 3-4 As shown. Figure 3 As shown, under reducing conditions, this monoclonal antibody specifically recognizes the heavy chain of tilapia IgM, with a size of approximately 75 kDa. Under non-reducing conditions, this monoclonal antibody can recognize both monomeric and polymeric forms of tilapia IgM, and even after the serum is diluted 500-fold, a clear IgM band can still be detected under both reducing and non-reducing conditions, indicating that this monoclonal antibody has high affinity and detection sensitivity. Furthermore, as... Figure 4 As shown, this monoclonal antibody can recognize tilapia IgM. + B cells indicate that the recognition epitope of this monoclonal antibody is located on the surface of the tilapia membrane-type IgM molecule.

[0089] Example 3: ELISA detection of serum IgM in tilapia

[0090] Serum from healthy tilapia was passed through a molecular sieve. Samples with elution volumes of 6-15 mL were collected for ELISA detection. The detection steps are as follows:

[0091] (1) The sample with elution position of 6-15 mL was diluted 10 times with coating buffer (0.05 M carbonate buffer, pH = 9.6) and added to the enzyme-labeled plate for coating, 100 μL per well, 4°C incubation overnight.

[0092] (2) After 5 times of washing with PBST (10 mM PBS, 0.05% Tween-20), 250 μL of PBST solution containing 5% skimmed milk was added to each well, and incubated at 37°C for 2 h.

[0093] (3) After 5 times of washing with PBST, the mouse anti-tilapia IgM monoclonal antibody was diluted to 2 μg / mL with PBST containing 5% skimmed milk as the primary antibody, 100 μL was added to each well, and incubated at 37°C for 1 h.

[0094] (4) After 5 times of washing with PBST, the HRP-labeled goat anti-mouse IgG (H+L) (Proteintech) antibody was diluted 1:2000 with PBST containing 5% skimmed milk as the secondary antibody, 100 μL was added to each well, and incubated at 37°C for 1 h.

[0095] (5) After 5 times of washing with PBST, 100 μL of TMB color developing solution was added to each well, and color developed at 37°C for 30 min in the dark.

[0096] (6) 50 μL of stop solution (2 M H2SO4) was added to each well to stop the reaction for 10 min, and then the absorbance value was detected at 450 nm wavelength with an enzyme-labeled instrument after mixing.

[0097] The detection results are shown in Table 2, which show that the mouse anti-tilapia IgM monoclonal antibody can be used for specific detection of tilapia natural IgM by ELISA, and has good specificity. Figure 5

[0098] In addition, the serum of tilapia was taken for dilution by half, and the highest dilution multiple of the mouse anti-tilapia IgM monoclonal antibody for detecting the serum of tilapia was explored by ELISA experiment, and the detection results are shown in Table 3, which show that the highest dilution multiple of the mouse anti-tilapia IgM monoclonal antibody for detecting the serum of tilapia is 51200 times.

[0099] Table 3 ELISA verification of the highest dilution multiple of the mouse anti-tilapia IgM monoclonal antibody for detecting the serum of tilapia

[0100]

[0101] Example 4 Affinity determination of monoclonal antibody

[0102] ​The present embodiment adopts surface plasmon resonance technology (SPR) to determine the antibody affinity, and the specific steps are as follows:

[0103] (1) According to the OpenSPR TM instrument standard operating procedure to install the COOH chip.

[0104] (2) Start running at the maximum flow rate (150 µL / min), and the detection buffer is PBST.

[0105] (3) After reaching the signal baseline, load 200 µL of IPA (isopropyl alcohol) and run for 10 s to exhaust the bubbles. After reaching the baseline, flush the sample ring with buffer and empty it with air.

[0106] (4) After the signal reaches the baseline, adjust the PBST flow rate to 20 µL / min.

[0107] (5) Load EDC / NHS (1:1) solution to activate the chip.

[0108] (6) Load 200 µL of Protein A affinity-purified Tilapia IgM diluted with PBST and run for 4 min, flush the sample ring, and empty it with air.

[0109] (7) Load 200 µL of Blocking solution, flush the sample ring with PBST, and empty it with air. Observe the baseline for 5 min to ensure stability.

[0110] (8) Dilute the antibody to be tested with PBS to 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, and 2.5 nM, respectively, and load 20 µL / min. The antigen and antibody binding time is 4 min; natural dissociation is 6 min.

[0111] (9) The binding and dissociation curve results are shown in Figure 6 , using the One To One analysis model, and using the TraceDrawer (Ridgeview Instruments ab, Sweden) software to analyze the affinity constant. The results show that the affinity constant of the mouse anti-Tilapia IgM monoclonal antibody can reach 6.02 × 10 -9 M, indicating that the antibody affinity is very strong.

[0112] Example 5: Analysis of the stability of hybridoma cell lines and the stability of secreted antibodies after subculture

[0113] The hybridoma cell strain OnM-14G10B1E1 was continuously subcultured to 25 generations, and the cells of the 0th, 5th, 10th, 15th, 20th and 25th generations were selected for amplification culture and collection of cell supernatant, and the total white blood cells of tilapia were taken for flow cytometry verification, and the serum of tilapia was taken for Western blot verification under reducing conditions. The verification results are shown in Figs. Figure 7 and Figure 8 As shown, the cell supernatants of the 0th, 5th, 10th, 15th, 20th and 25th generations have no difference in recognition of tilapia IgM + B cells and secreted IgM in serum, indicating that the hybridoma cell strain has subculture stability and antibody secretion stability after subculture.

[0114] Example 6: Determination of nucleic acid sequences of heavy chain variable region and light chain variable region of monoclonal antibody

[0115] The OnM-14G10B1E1 hybridoma cells in logarithmic growth phase were taken, total RNA was extracted by Trizol (TAKARA), and cDNA was reversely transcribed by using oligo (dT) 20 (Invitrogen) as a primer; then the heavy chain variable region and light chain variable region genes were amplified by using specific PCR primers with cDNA as a template; the PCR products were recovered and purified after agarose gel electrophoresis, and then were connected into pMD-18T vector by TA cloning, followed by sequencing and sequence analysis, to obtain the coding gene of the heavy chain variable region as shown in SEQ ID NO. 9, encoding the protein as shown in SEQ ID NO. 7; and the coding gene of the light chain variable region as shown in SEQ ID NO. 10, encoding the protein as shown in SEQ ID NO. 8. The CDR region (CDRH1, CDRH2, CDRH3) of the heavy chain variable region comprises the sequences as shown in SEQ ID NO. 1-3, respectively, and the CDR region (CDRL1, CDRL2, CDRL3) of the light chain variable region comprises the sequences as shown in SEQ ID NO. 4-6, respectively.

[0116] In summary, the present application successfully screens a hybridoma cell strain OnM-14G10B1E1 which can specifically secrete mouse anti-tilapia IgM monoclonal antibody, and the hybridoma cell strain is stable in subculture and antibody secretion, and the mouse anti-tilapia IgM monoclonal antibody secreted by the hybridoma cell strain is prepared, which can specifically recognize secreted IgM and IgM + B cells of tilapia, and has the characteristics of good specificity, high titer, sensitive reaction and strong affinity, and is of great significance for the research of immune system of tilapia and the evaluation of immune prevention technology of diseases.

[0117] The above detailed description describes the implementation of the present application, but the present application is not limited to the specific details in the above implementation. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A monoclonal antibody against tilapia IgM, characterized in that, The sequences of the heavy chain variable regions CDRH1, CDRH2, and CDRH3 of the monoclonal antibody are shown in SEQ ID NO.1-3, respectively, and the sequences of the light chain variable regions CDRL1, CDRL2, and CDRL3 of the monoclonal antibody are shown in SEQ ID NO.4-6, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. A polynucleotide encoding the monoclonal antibody of any one of claims 1-2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 3.

5. A host cell, characterized in that, The host cell contains the polynucleotide of claim 3 or the recombinant vector of claim 4.

6. A hybridoma cell line OnM-14G10B1E1, characterized in that, The hybridoma cell line OnM-14G10B1E1 has the accession number CCTCC NO: C2023153.

7. The use of the monoclonal antibody of any one of claims 1-2, the polynucleotide of claim 3, the recombinant vector of claim 4, the host cell of claim 5, or the hybridoma cell line of claim 6 in the preparation of a tilapia IgM detection kit.

8. The application according to claim 7, characterized in that, The detection kits include: colloidal gold immunoassay kits, chemiluminescence kits, enzyme-linked immunosorbent assay kits, or immunofluorescence kits.

9. A kit for detecting IgM in tilapia, characterized in that, The kit contains any of the monoclonal antibodies of claims 1-2 or the hybridoma cell line of claim 6.

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