A mouse anti-carassius auratus igm monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The present application relates to a kind of mouse anti-carassius auratus IgM monoclonal antibody, hybridoma cell strain and its application.The mouse anti-carassius auratus IgM monoclonal antibody is obtained by the hybridoma cell strain CaJYM-12H6F2G4C6 with the preservation number of CCTCC NO:C2024233 secretion.The monoclonal antibody can be used for Western blot and ELISA specific detection carassius auratus secretory form IgM, and can be used for flow cytometry identification carassius auratus IgM + B cell.This monoclonal antibody is specific, high sensitivity, can be used for the structural analysis of carassius auratus IgM, immune response level detection etc., lays a foundation for the in-depth study of carassius auratus immune system and the establishment of antibody level as index vaccine immune effect evaluation method.

Description

TECHNICAL FIELD

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

[0002] Carassius auratus belongs to Cypriniformes, Cyprinidae and Carassius, and is widely favored by consumers due to its delicious taste, tender meat quality and rich nutrition. The China Fishery Statistical Yearbook shows that the annual production of Carassius auratus in China has exceeded 270 million tons for many consecutive years, and Carassius auratus is one of the important freshwater economic fish species in China. As is known to all, vaccination can activate the fish immune system and improve the disease resistance of the body against specific pathogens, which is an important means of preventing Carassius auratus diseases and is also 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 tedious, time-consuming and lacks evaluation standards. Based on the monoclonal antibody of fish immunoglobulin (Ig), the change rule of the level of specific antibody (i.e. secreted Ig) in fish vaccinated with the vaccine is analyzed to evaluate the immune effect of the vaccine, which will provide a powerful tool for establishing the evaluation standard system of the immune effect of the vaccine of Carassius auratus.

[0003] As the earliest discovered and the most abundant immunoglobulin in teleosts, IgM has been proved to play a very important immune function in the systemic immunity and mucosal immunity of fish. Therefore, the development of the monoclonal antibody of Carassius auratus IgM is helpful to deeply understand the humoral immune response rule of Carassius auratus, and can help to establish the antibody analysis and detection method of Carassius auratus, and then establish the method for evaluating the immune effect of the vaccine based on the antibody level of Carassius auratus and formulate the use regulations of the vaccine, so as to promote the development of the immunoprophylaxis technology of the infectious diseases of Carassius auratus taking vaccination as the core. SUMMARY

[0004] The application provides a hybridoma cell strain CaJYM-12H6F2G4C6, which is obtained by immunizing mice with natural IgM of Carassius auratus serum purified by Protein A affinity and screening a large number of times. The hybridoma cell has been preserved in the China Center for Type Culture Collection on July 16, 2024, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C2024233.

[0005] The application further provides a mouse anti-Carassius auratus IgM monoclonal antibody, which is secreted by the hybridoma cell with the preservation number of CCTCC NO: C2024233.

[0006] Further, the CDR of the heavy chain variable region of the monoclonal antibody is the following three sequences:

[0007] HYEMS (CDRH1, SEQ ID NO. 1),

[0008] WMNIYTGEPTYADDFKG (CDRH2, SEQ ID NO. 2),

[0009] WIYGSSGWFFDV (CDRH3, SEQ ID NO. 3)

[0010] Further, the CDRs of the light chain variable region of the monoclonal antibody are the following three sequences:

[0011] RSSQNIIHSNGNTYLE (CDRL1, SEQ ID NO. 4),

[0012] KVSNRFF (CDRL2, SEQ ID NO. 5),

[0013] FQGSHVPYT (CDRL3, SEQ ID NO. 6).

[0014] The amino acid sequence of the heavy chain variable region of the above-mentioned monoclonal antibody is: MAWVWTLLFLMAAAQSVQAQIQLVQSGPELKKPGETVKISCKASGYTFTHYEMSWMKQAPGKGLKWMGWMNIYTGEPTYADDFKGRFVFSLETSASTAYLHINNLKNEDTATYFCARWIYGSSGWFFDVWGAGTTVTVSS (SEQ ID NO. 7); preferably, the polynucleotide encoding the amino acid is: ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTGTCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACACACTATGAAATGAGCTGGATGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATGAACATTTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGTCGGTTTGTCTTCTCTTTGGAAACCTCAGCCAGCACTGCCTATTTGCACATCAACAACCTCAAAAATGAGGACACGGCTACGTATTTCTGTGCAAGATGGATCTACGGTAGTAGCGGCTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO. 8);

[0015] The amino acid sequence of the light chain variable region is: MKLPVRLLVLMFWIPASSSDVLMTQSPLSLPVGLGDQASISCRSSQNIIHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFFGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK (SEQ ID NO. 9); preferably, the polynucleotide encoding the amino acid is: ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCAGTCGGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCCAGTCAGAACATTATACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTTTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA (SEQ ID NO. 10).

[0016] The present application also provides a polynucleotide encoding the monoclonal antibody, or encoding the CDR region, or encoding the variable region of the heavy chain and the light chain.

[0017] The present application also provides a recombinant vector containing the polynucleotide, and a host cell containing the polynucleotide or the recombinant vector.

[0018] The present application also provides the use of the hybridoma cell strain, the monoclonal antibody, the polynucleotide, the recombinant vector, or the host cell in the preparation of a detection kit for crucian IgM.

[0019] Further, the detection kit is an ELISA detection kit.

[0020] The present application provides a kit for the detection of crucian IgM, which comprises the hybridoma cell strain and / or the monoclonal antibody.

[0021] Beneficial effects: the application adopts Protein A affinity purification of crucian serum natural IgM as antigen to immunize mice and screen to obtain a hybridoma cell strain CaJYM-12H6F2G4C6 capable of specifically secreting mouse anti-crucian IgM monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell strain can specifically recognize crucian secreted form IgM and IgM + B cells, and is specific, high in titer, sensitive in reaction, and can be used for structure analysis of crucian IgM, detection of immune response level and the like. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below, and it should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 It is a result map of SDS-polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions for detecting purified crucian serum natural IgM and staining with coomassie brilliant blue.

[0024] Figure 2 It is a result map of ELISA analysis of mouse anti-crucian IgM monoclonal antibody subclass.

[0025] Figure 3 It is a result map of Western blot under reducing and non-reducing conditions for detecting crucian serum IgM bands after being diluted by 100 and 500 times respectively by using mouse anti-crucian IgM monoclonal antibody.

[0026] Figure 4 It is a result map of flow cytometry for detecting IgM + B cells in the kidney tissue of crucian.

[0027] Figure 5 It is a result map of Western blot and ELISA analysis of mouse anti-crucian IgM monoclonal antibody for detecting IgM TM 200 Increase 10 / 300 GL (GE Healthcare) after separation in different elution volumes.

[0028] Figure 6To verify the recognition of supernatant of 0th, 5th, 10th, 15th and 20th generation cells of CaJYM-12H6F2G4C6 hybridoma cell strain by flow cytometry, the recognition of supernatant of 0th, 5th, 10th, 15th and 20th generation cells of CaJYM-12H6F2G4C6 hybridoma cell strain on IgM in kidney of Carassius auratus was verified by flow cytometry. + The recognition result of B cells is shown in the figure.

[0029] Figure 7 To verify the recognition of supernatant of 0th, 5th, 10th, 15th and 20th generation cells of CaJYM-12H6F2G4C6 hybridoma cell strain by Western blot, the recognition result of supernatant of 0th, 5th, 10th, 15th and 20th generation cells of CaJYM-12H6F2G4C6 hybridoma cell strain on secreted IgM in serum of Carassius auratus was verified by Western blot. DETAILED DESCRIPTION

[0030] The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore are only examples and cannot be used to 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 commonly understood meanings 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.

[0031] Example 1 Preparation of a mouse anti-Carassius auratus IgM monoclonal antibody

[0032] In this example, HiTrap® Protein A column (GE Healthcare) was used to purify natural IgM protein in serum of Carassius auratus, and the purified natural IgM protein was used as an antigen to immunize mice to obtain hybridoma cell strain CaJYM-12H6F2G4C6, and the mouse anti-Carassius auratus IgM monoclonal antibody was produced by the hybridoma cell strain, and the specific process was as follows:

[0033] (1) Obtaining serum of Carassius auratus

[0034] A healthy Carassius auratus was immersed in 30 mg / L fish anesthetic MS-222 (Sigma), and after anesthesia, it was placed in a dissection tray, the head was covered with a towel, the scales on the lateral line of the posterior margin of the anal fin were removed, and a 5 mL syringe without anticoagulant was inserted into the muscle tissue at the posterior of the anal fin on the midline until the needle reached the spine, and the blood entered the syringe; after the blood was coagulated, it was placed at 4°C overnight, and the next day it was centrifuged at 4°C, 3000 g for 10 min, and the upper serum was transferred to a clean centrifuge tube.

[0035] (2) Purification of natural IgM in serum of Carassius auratus

[0036] ① Equilibrate the column: rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS, pH=7.2).

[0037] ② Loading: The carp serum was centrifuged at 4℃, 15000 rpm / min for 30 min to remove impurities, and then filtered through a 0.22 μm filter. The serum was diluted 1:3 with Binding buffer and then loaded onto a Protein A affinity purification column through an AKTA pure 25L protein purification system (GE Healthcare).

[0038] ③ Neutral washing: The Protein A affinity purification column was washed with 20 mL of Binding buffer to remove unbound impurities.

[0039] ④ Acid elution: The Protein A affinity purification column was washed with 0.1 M citric acid solution at pH=3.0, and the eluate was collected in a centrifuge tube pre-filled with 200 μL of 1 mol / L Tris-HCl solution at pH=9.0.

[0040] ⑤ The collected eluate was subjected to SDS-PAGE to detect the purity of the purified carp IgM. The detection results are shown in FIG. 1. Under reducing conditions, it was a heavy chain (70-100 kDa) and a light chain (25-35 kDa), and under non-reducing conditions, it was a monomer or a multimer greater than 180 kDa, with a purity of 91%. Figure 1

[0041] ⑥ The purified carp serum IgM was diluted with PBS at pH=7.4 and ultrafiltrated, and finally the protein concentration was determined using a BCA kit (Biosharp).

[0042] (3) Mouse immunization

[0043] Five 6-8 week old BALB / c mice were immunized with the purified carp serum natural IgM in step (2) as an immunogen. The immunization route and procedure were as follows: about 50 μg of carp serum natural IgM was mixed and emulsified with an equal volume of Freund's complete adjuvant, and was injected subcutaneously in multiple points on the back of the mouse. Two weeks later, the mouse was boosted three times at two-week intervals. The antigen protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and was injected subcutaneously in multiple points on the back of the mouse. On the 7th day after the third boost, tail vein blood was collected for ELISA detection of antibody titer.

[0044] (4) Cell fusion and culture

[0045] ​① Cell fusion: the spleen cells of the immunized mouse in step (3) were mixed with mouse myeloma cells SP2 / 0 (ATCC) at a cell ratio of 5:1, centrifuged at 1000 rpm / min for 10 min, and the supernatant was discarded. 1 mL PEG-1450 (Sigma) was slowly added to the test tube while shaking; then 40 mL complete medium was slowly added for several times. The whole process was carried out in a 37°C water bath, followed by centrifugation at 1000 rpm / min for 10 min, and the supernatant was removed to complete the cell fusion.

[0046] ② Cell culture: the cell clumps after centrifugation were gently dispersed, resuspended with HAT medium, and plated in the prepared 96-well plate containing feeder cells at 200 μL per well. It was placed in a 37°C, 5% CO2 incubator for culture. After one week, the growth of 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 bottom, the culture supernatant was removed for ELISA detection.

[0047] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies

[0048] ① Screening of positive clones: the purified crucian serum natural IgM (2 μg / mL) was used to coat the ELISA plate, which was incubated at 4°C overnight. The next day, after washing and blocking, 100 μL of the culture supernatant of the hybridoma cells to be detected 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 terminate the reaction. The absorbance was detected at 450 nm wavelength by a microplate reader. The absorbance value greater than 2 times that of the negative control was considered positive.

[0049] ② Subcloning of hybridoma cells: the hybridoma cells positive in ELISA were gently eluted from the culture plate, counted, and diluted with culture medium to 100 μL per well in a 96-well plate. After 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, and the results of the third screening are shown in Table 1. After repeated screening, the screening conditions were: high antibody titer, high antibody concentration, and stable secretion in the supernatant of the cells detected by ELISA, and the monoclonal antibody could recognize the monomer or multimer form of crucian IgM, and could be used for flow cytometry to recognize crucian IgM + B cells.

[0050] Finally, a hybridoma cell strain stably expressing anti-Goldfish IgM monoclonal antibody was obtained, which was named hybridoma cell strain CaJYM-12H6F2G4C6, and was preserved in China Center for Type Culture Collection on July 16, 2024, located in Wuhan University, Wuhan, China, with a preservation number of CCTCC NO: C2024233.

[0051] Table 1: Results of supernatant ELISA and flow cytometry detection of the third subcloning cell strain

[0052]

[0053] ③Mass production of monoclonal antibody: 3 healthy BALB / c mice about 10 weeks old were injected intraperitoneally with liquid paraffin, 0.5 mL per mouse, and 1 week later, each pretreated mouse was injected intraperitoneally with 5×10 6 The ascites were extracted when the mouse abdomen was extremely swollen after 10-14 days, once every 2 days, a total of 6.8 mL of ascites. The extracted ascites were centrifuged at 18000 g for 10 min at 4℃, and the supernatant was subjected to affinity chromatography purification, and a total of 12.9 mg of purified product was obtained, which was the murine anti-Goldfish IgM monoclonal antibody. The concentration of murine anti-Goldfish IgM monoclonal antibody in ascites was 1.89 mg / mL, and the titer was 1:2048000 (as shown in Table 2).

[0054] Table 2: Results of murine anti-Goldfish IgM monoclonal antibody titer determination

[0055]

[0056] Note: The antibody titer evaluation criteria are as follows: if the OD of the test well is greater than 0.1 and greater than 2.1 times that of the negative control well (P / N>2.1, where 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 of the antibody determined to be positive is the antibody titer.

[0057] Example 2: Identification of murine anti-Goldfish IgM monoclonal antibody

[0058] (1) Detection of antibody subclass, process as follows:

[0059] The purified crucian serum natural IgM protein is coated into an ELISA plate, incubated at 4°C overnight, washed with PBST for 3 times, then 100 μL of mouse anti-crucian IgM monoclonal antibody with a final concentration of 1 μg / mL is added to each well, and incubated at 37°C for 2 h; after washing with PBST for 3 times, 100 μL of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA and IgM monoclonal antibody diluted is added to each well, and incubated at 37°C for 1 h; after washing for 3 times, 100 μL of TMB color developing liquid (Bi Yun Tian) is added to each well, and incubated at 37°C for 30 min in the dark; then 50 μL of stop solution (2 M H2SO4) is added to each well to terminate the reaction. The absorbance value is detected by an enzyme-labeled instrument at a wavelength of 450 nm, and the HRP-labeled goat anti-mouse Ig subclass used in the positive reaction well is used as the antibody class to be detected. The identification result is shown in Figure 2 Fig. 1, which shows that the mouse anti-crucian IgM monoclonal antibody prepared in the application is IgG1.

[0060] (2) Western blot detection, the process is as follows:

[0061] ① Preparation of sample: the serum of healthy crucian is diluted by 100 and 500 times with PBS respectively, then 1 / 5 volume of 5x reduced and non-reduced SDS-PAGE loading buffer (Solarbio) is added, vortexed, heated at 100°C for 10 min in a metal bath, and placed on ice for standby or stored at -80°C.

[0062] ② SDS-PAGE: 12% and 8% Tris-glycine polyacrylamide lower separation gel and 5% upper concentrated gel are prepared respectively, and then electrophoresis is performed, and the electrophoresis conditions are as follows: 80 V, 0.5 h; 120 V, 1 h.

[0063] ③ Membrane transfer: the SDS-PAGE gel is 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 is cut to an appropriate size, soaked in methanol for 30 s for activation, then transferred into the membrane transfer buffer for soaking, then stacked in the order of filter paper-PVDF membrane-gel-filter paper from bottom to top, ensuring that there are no air bubbles in each layer, and the proteins on the SDS-PAGE gel are transferred to the PVDF membrane at 300 mA for 60 min.

[0064] ④ Blocking: after the membrane transfer is completed, the PVDF membrane is transferred into 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.

[0065] (5) Primary antibody incubation: Discard the blocking solution, dilute the mouse anti-Carassius auratus IgM monoclonal antibody to 2 μg / mL with TBST solution containing 5% skimmed milk powder, and incubate at 4°C overnight.

[0066] (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 (Proteintech) with TBST solution containing 5% skimmed milk powder at 1:5000, and incubate at room temperature for 1 h.

[0067] (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 solutions at a ratio of 1:1, drop the color developing solution onto the PVDF membrane, and after sufficient contact, analyze and take a photo using a chemiluminescence imager.

[0068] (3) Flow cytometry detection, the process is as follows:

[0069] (1) Carassius auratus head kidney tissue total white blood cell separation: Anesthetize healthy Carassius auratus, cut the body cavity of Carassius auratus from the cloaca, take out the head kidney tissue and place it in DMEM medium (Gibco), cut it into 1 mm 3 tissue fragments with scissors, then transfer it to a 100 µm cell sieve (Biosharp), gently grind the tissue fragments with a 2 mL syringe piston, slowly add DMEM medium during the grinding process, so that single cells pass through the 100 µm cell sieve, and prepare a single cell suspension of the head kidney tissue; then add the single cell suspension to the 34% and 51% discontinuous Percoll (GE Healthcare) liquid surface, and after centrifugation, the white blood cells between the two layers of discontinuous Percoll are the total white blood cells of the head kidney tissue of Carassius auratus.

[0070] (2) Primary antibody incubation: wash the total white blood cells of the head kidney tissue of Carassius auratus with PBS containing 2% FBS (Yeasen) twice, then add the mouse anti-Carassius auratus IgM monoclonal antibody, with a final concentration of 2 μg / mL, and incubate on ice for 45 min, gently vortex the cells once every 15 min.

[0071] (3) Secondary antibody incubation: wash the cells with PBS containing 2% FBS twice, then add PE Goat Anti-Mouse IgG (Biolegend), with a final concentration of 1 μg / mL, and incubate on ice for 30 min, gently vortex the cells once every 10 min.

[0072] (4) Flow detection: wash the cells with PBS containing 2% FBS twice, resuspend the cells and filter them, then use a flow cytometer (BD) to detect the proportion of IgM + B cells in the lymphocytes of the head kidney tissue of Carassius auratus.

[0073] The mouse anti-Carassius auratus IgM monoclonal antibody can recognize the secreted form of IgM and IgM + The recognition and specificity verification of B cells are as shown in Figure 3-4 Under reducing conditions, the monoclonal antibody recognizes the heavy chain of Carassius auratus IgM with a size of 70-100 kDa; under non-reducing conditions, the monoclonal antibody can recognize the monomeric or polymeric form of Carassius auratus IgM. In addition, the monoclonal antibody can recognize the membrane form of Carassius auratus IgM + B cells, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of the membrane form of Carassius auratus IgM molecule. Sequencing shows that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 9, wherein the CDR region (CDRH1, CDRH2, CDRH3) of the heavy chain variable region comprises the sequences shown in SEQ ID NO. 1-SEQ ID NO. 3, respectively, and the CDR region (CDRL1, CDRL2, CDRL3) of the light chain variable region comprises the sequences shown in SEQ ID NO. 4-SEQ ID NO. 6, respectively.

[0074] Example 3: ELISA detection of Carassius auratus serum IgM using the monoclonal antibody provided by the present application

[0075] After 2 mL of healthy Carassius auratus serum was centrifuged at 18,000 g for 30 min, impurities were removed through a 0.22 μm filter membrane, and then the serum sample was loaded onto a Superdex TM 200Increase 10 / 300 GL column (GE Healthcare) for gel filtration chromatography, and samples with elution volumes of 6-15 mL were taken for Western bolt and ELISA detection, respectively. The Western bolt detection method is as described in step (2) of Example 2, and the ELISA detection method is as follows:

[0076] (1) Coating: The samples with elution volumes of 6-15 mL were diluted 10 times with coating buffer (0.05 M carbonate buffer, pH=9.6), and 100 μL of the diluted sample was coated in each well of the enzyme-labeled plate, which was incubated at 4°C overnight.

[0077] (2) Blocking: After washing 5 times with PBST (10 mM PBS, 0.05% Tween-20), 250 μL of PBST solution containing 5% skimmed milk powder was added to each well, and incubated at 37°C for 2 h.

[0078] (3) Primary antibody incubation: After washing 5 times with PBST, the mouse anti-crucian IgM monoclonal antibody was diluted to 1 μg / mL with PBST solution containing 5% skim milk powder as the primary antibody. 100 μL was added to each well and incubated at 37℃ for 1 h.

[0079] (4) Secondary antibody incubation: After washing 5 times with PBST, dilute HRP-labeled goat anti-mouse IgG (H+L) (Proteintech) antibody with PBST solution containing 5% skim milk powder at a ratio of 1:5000 as secondary antibody, add 100 μL to each well, and incubate at 37℃ for 1 h.

[0080] (5) Color development: After washing with PBST 5 times, add 100 μL of TMB color development solution to each well and develop the color at 37℃ in the dark for 30 min.

[0081] (6) Termination reading: Add 50 μL of stop solution (2 M H2SO4) to each well to terminate the reaction for 10 min. After mixing, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm.

[0082] Test results as follows Figure 5 As shown, ELISA detects OD 450 The magnitude of the value corresponds to the band signal intensity detected by Western blot, indicating that the mouse anti-crucian IgM monoclonal antibody can be used for ELISA to specifically detect natural IgM in crucian carp, with good specificity.

[0083] In addition, crucian carp serum was serially diluted, and the highest dilution factor at which the mouse anti-crucian carp IgM monoclonal antibody could detect crucian carp serum was investigated by ELISA. The results are shown in Table 3, indicating that the highest dilution factor at which the mouse anti-crucian carp IgM monoclonal antibody could detect crucian carp serum was 25,600 times.

[0084] Table 3. ELISA validation of the highest dilution factor of mouse anti-crucian carp IgM monoclonal antibody that can detect crucian carp serum.

[0085]

[0086] Example 4: Stability analysis of hybridoma cell lines after passage and stability analysis of secreted antibodies after passage

[0087] Hybridoma cell line CaJYM-12H6F2G4C6 was continuously passaged to 20 generations. Cells from generations 0, 5, 10, 15, and 20 were selected for amplification culture, and cell supernatants were collected. Total leukocytes from crucian carp head kidney tissue were used for flow cytometry verification, and crucian carp serum was used for Western blot verification under reducing conditions. The verification results are as follows: Figure 6 and Figure 7As shown, the results show that the supernatant of the 0th, 5th, 10th, 15th and 20th generation of cells can specifically recognize the IgM of the crucian carp + The B cells and the secreted form IgM in serum have no difference in recognition, which indicates that the hybridoma cell strain has the stability in generation and the stability in antibody secretion after generation.

[0088] Example 5: Determination of the nucleic acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody

[0089] The hybridoma cells of CaJYM-12H6F2G4C6 in the logarithmic growth phase are taken, the total RNA is extracted by using the Trizol (TAKARA) method, the cDNA is reversely transcribed by using oligo(dT)20 (Invitrogen) as a primer, the heavy chain variable region and the light chain variable region genes are amplified by using specific PCR primers respectively, the PCR products are recovered and purified after agarose gel electrophoresis, the pMD-18T vector is connected by using TA cloning, and then sequencing and sequence analysis are performed, the coding gene sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the protein shown as SEQ ID NO. 7 is encoded; the coding gene sequence of the light chain variable region is shown as SEQ ID NO. 10, and the protein shown as SEQ ID NO. 9 is encoded.

[0090] In summary, the mouse anti-crucian carp IgM monoclonal antibody is successfully prepared by the hybridoma cell technology, the monoclonal antibody can specifically recognize the crucian carp IgM, the monoclonal antibody can be used for detecting the secreted form IgM and IgM + The B cells, the secreted form IgM and the secreted form IgM

[0091] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be variously modified and changed, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A hybridoma cell line CaJYM-12H6F2G4C6, characterized in that, The preservation number of the hybridoma cell strain CaJYM-12H6F2G4C6 is CCTCC NO: C2024233.

2. The monoclonal antibody secreted by the hybridoma cell strain of claim 1.

3. The monoclonal antibody according to claim 2, characterized in that, 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~ SEQ ID NO. 3, and the sequences of CDRL1, CDRL2 and CDRL3 of the light chain variable region are respectively shown as SEQ ID NO. 4~ SEQ ID NO.

6.

4. The monoclonal antibody of claim 2, wherein, 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.

9.

5. A polynucleotide encoding the monoclonal antibody of any one of claims 2-4.

6. A recombinant vector, characterized in that, The polynucleotide of claim 5.

7. A host cell, characterized in that, The polynucleotide of claim 5 or the recombinant vector of claim 6.

8. Use of the hybridoma cell strain of claim 1, the monoclonal antibody of any one of claims 2-4, the polynucleotide of claim 5, the recombinant vector of claim 6 or the host cell of claim 7 in the preparation of a carp IgM detection kit.

9. Use according to claim 8, characterized in that, The detection kit is an ELISA detection kit.

10. A kit for detecting the Crassostrea hongkongensis IgM, characterized in that, The kit comprises the hybridoma cell strain of claim 1 and / or the monoclonal antibody of any one of claims 2-4.

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