Anti-zebra fish IgM monoclonal antibody and preparation and application thereof

Abstract

The invention discloses an anti-zebra fish IgM monoclonal antibody and preparation and application thereof, and belongs to the technical field of monoclonal antibodies, and the anti-zebra fish IgM monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CCTCC NO: C202542. The anti-zebrafish IgM monoclonal antibody provided by the invention not only can specifically recognize IgM in serum and IgM + B cells in tissues, but also can be used for evaluating the immune effect of vaccines, is wide in application, and has positive significance on scleroderma immunity and vaccine research.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to an anti-zebrafish IgM monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Zebrafish, as an internationally recognized vertebrate model, are characterized by rapid reproduction, numerous offspring, and a high degree of genetic similarity to humans. They are not only valuable for studying the immune mechanisms of bony fishes and playing a crucial role in disease control during aquaculture, but also for researching vertebrate development, evolution, genetics, and disease, providing a unique perspective for understanding the evolution, development, and function of the entire vertebrate immune system. Therefore, zebrafish are an ideal model for studying the basic immunology of bony fishes and even vertebrates in general. However, zebrafish have relatively few immune-related antibodies. Research on zebrafish Ig monoclonal antibodies is crucial for gaining a deeper understanding of the immune response patterns in zebrafish and for elucidating the immune responses of bony fishes.

[0003] Immunoglobulins (Igs) are the main molecules mediating the body's immunity. They are produced by plasma cells that differentiate from B cells and can specifically bind to corresponding antigens. A typical Ig molecule consists of two heavy chains (IgH) linked by disulfide bonds and two light chains (IgL). Immunoglobulins expressed on the surface of B cells are usually described as B cell receptors (BCRs), while secreted Igs are described as antibodies. In bony fishes, three major Ig isotypes (IgM, IgD, and IgT / IgZ) have been identified. IgM is a key effector molecule in the immune system of bony fishes and was the first immunoglobulin identified in bony fishes. Among all immunoglobulins, IgM is absolutely dominant in all immune tissues and plays a very important role in the body's resistance to pathogens.

[0004] Most commercially important fish species currently possess IgM monoclonal antibodies, and their immunizing antigens are mostly natural IgM purified from serum. However, zebrafish are small in size and have too little serum, making it impossible to obtain purified natural IgM as an antigen. Therefore, there is currently no monoclonal antibody that can accurately identify zebrafish IgM. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides an anti-zebrafish IgM monoclonal antibody, its preparation method and application, providing antibody materials for immune research and vaccine evaluation in bony fish.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a hybridoma cell line that secretes a monoclonal antibody against zebrafish IgM. This hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China, on November 19, 2025, with accession number CCTCC NO:C202542.

[0007] The present invention obtains the above-mentioned hybridoma cell line by the following method: using the fusion protein IgM-CH2-CH4 as an immunogen, female BALB / c mice are immunized to fuse the spleen cells and myeloma cells of the immunized mice, and then obtained through a large number of screenings; wherein, the fusion protein IgM-CH2-CH4 contains the amino acid sequence shown in SEQ ID NO.1, and the fusion protein IgM-CH2-CH4 can be specifically prepared by expression in 293F cells.

[0008] Secondly, the present invention provides an anti-zebrafish IgM monoclonal antibody, which is secreted by a hybridoma cell line with accession number CCTCCNO:C202542.

[0009] Thirdly, the present invention provides a method for preparing anti-zebrafish IgM monoclonal antibody, namely, culturing a hybridoma cell line with accession number CCTCC NO:C202542 in vivo or in vitro, and then purifying it to obtain the antibody.

[0010] In some embodiments, the above method specifically involves: injecting a hybridoma cell line into a mouse via intraperitoneal injection, extracting ascites fluid 10-14 days later, centrifuging the fluid, and then purifying the supernatant.

[0011] Fourthly, the present invention provides applications of the above-mentioned anti-zebrafish IgM monoclonal antibody or reagents / kits containing the antibody, including at least the following: i) Application in the specific detection of IgM in zebrafish serum; ii) IgM in zebrafish tissue + Applications in B cell-specific detection; iii) Application in evaluating the immunization effect of vaccines.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes the CH2-CH4 region of IgM expressed in eukaryotic 293F cells, and uses the resulting protein as an antigen to immunize mice. After cell fusion, hybridoma cells are screened for monoclonal antibodies, yielding a monoclonal antibody that accurately recognizes zebrafish IgM. The antibody provided by this invention can specifically recognize IgM in serum and tissues. + B cells can also be used to evaluate the effectiveness of vaccines and have a wide range of applications.

[0013] Preservation of biological materials in this invention: Preservation material: hybridoma cell line (zebrafish); Deposit date: November 19, 2025; Preservation institution: China Center for Type Culture Collection (CCTCC); Accession number: CCTCC NO:C202542. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0015] Figure 1 This is a map of the eukaryotic expression plasmid constructed in Example 1; Figure 2 This is a graph showing the detection results of the recombinant protein obtained after purification in Example 1; Figure 3 Wild-type zebrafish (wt) and IgM knockout zebrafish (IgM) were used in Example 3. - / - Western blot results of serum; Figure 4 The results of flow cytometry analysis of head kidney tissue from wild-type zebrafish and IgM knockout zebrafish in Example 3; Figure 5 For example 3, the marker genes for B cells and T cells in IgM + and IgM - Analysis of expression in cell populations, ns indicates no significance, * indicates p <0.05, ** indicates p <0.01; Figure 6 The results of immunofluorescence of the spleens of wild-type zebrafish and IgM knockout zebrafish in Example 3; Figure 7 This is the verification result of the mouse anti-zebrafish IgM monoclonal antibody in Example 4 recognizing specific IgM against Streptococcus agalactiae in zebrafish serum. Detailed Implementation

[0016] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0018] Zebrafish are an ideal model for studying the basic immunology of bony fishes and even vertebrates. Research on zebrafish Ig (especially IgM) monoclonal antibodies is helpful for a deeper understanding of the immune response patterns in bony fishes. However, there is currently a lack of zebrafish IgM monoclonal antibodies, and related research is limited. To address the technical problem of the lack of zebrafish IgM monoclonal antibodies, this invention provides an anti-zebrafish IgM monoclonal antibody prepared from a hybridoma cell line with accession number CCTCC NO: C202542.

[0019] Furthermore, in this embodiment of the invention, an IgM-CH2-CH4-pIL2-1-hFc-N eukaryotic expression vector is constructed, utilizing 293F cells to express the CH2-CH4 region of IgM in eukaryotic cells. Simultaneously, the design of the N-terminal signal peptide ensures the effective secretion of the target protein into the extracellular supernatant, thereby obtaining a large quantity of high-purity protein. In addition, the presence of the Fc tag improves the stability of the entire fusion protein, reducing degradation and aggregation during storage and handling. The recombinant IgM protein obtained by this method is active, structurally closer to the natural protein, and has a higher protein concentration. Then, mice are immunized with the above-mentioned recombinant IgM protein. After cell fusion, the hybridoma cells are screened for monoclonal antibodies, yielding the aforementioned hybridoma cell line. Finally, the hybridoma cell line can be used to mass-produce monoclonal antibodies against zebrafish IgM.

[0020] Experimental data show that the anti-zebrafish IgM monoclonal antibody provided by this invention can specifically recognize IgM in serum and tissues. + B cells, and do not recognize IgM - / - In addition, the antibody can recognize specific IgM in the serum of zebrafish infected with Streptococcus agalactiae, which lays the foundation for the establishment of a vaccine immunization efficacy evaluation method based on antibody levels in bony fishes.

[0021] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0022] Example 1 This example provides a zebrafish IgM antigen containing the amino acid sequence shown in SEQ ID NO.1, and its preparation method is as follows: like Figure 1 As shown, an IgM-CH2-CH4-PTT5 eukaryotic expression vector was constructed, with a signal peptide added to the N-terminus to guide secretory expression. An hFc tag was added to the posterior end of the signal peptide to facilitate purification. Mammalian 293F suspension cells were used as the host for recombinant expression.

[0023] The column was packed with protein A and equilibrated with equilibration buffer (20 mM Na2HPO4, pH 7.4). The expression supernatant containing the recombinant protein, filtered through a 0.45 μm filter, was then incubated with protein A / G for 1 h. The column was washed with 5-10 column volumes of equilibration buffer, and the Fc-tagged protein was eluted with elution buffer (0.1 M glycine, pH 2.5). The protein pH was neutralized to 7.4 with 1 M Tris. The purified protein bands were detected using Coomassie Brilliant Blue. Results are as follows: Figure 2 As shown.

[0024] Example 2 Using the IgM antigen prepared in Example 1, this invention provides a mouse anti-zebrafish IgM monoclonal antibody, the preparation process of which is as follows: (1) Immunize mice.

[0025] Healthy female BALB / c mice aged 7-8 weeks were immunized three times with a two-week interval between each immunization. The first immunization was given with Freund's complete adjuvant, and the second and third immunizations were given with Freund's incomplete adjuvant. Blood samples were collected after the second immunization to detect antibody production.

[0026] (2) Cell fusion.

[0027] Feeder cells were prepared 3-5 days before fusion. Peritoneal macrophages from BALB / c mice were seeded into 96-well cell culture plates. Antibody-positive mice were euthanized by cervical dislocation, and spleen cells were aseptically harvested. The spleen cells were fused with SP2 / 0 myeloma cells at a ratio of 5:1 using PEG-4000. The fused cells were then seeded onto the prepared feeder cells.

[0028] (3) Screening and cloning of positive hybridoma cell lines. Polystyrene plates were coated with purified zebrafish IgM, and the cell culture supernatant was collected on days 10-15 after fusion for indirect ELISA detection. The positive hybridoma cells were expanded and cultured, and the positive hybridoma cells were cloned using the limiting dilution method. At least three rounds of cloning were performed, and the cloned positive hybridoma cells were cryopreserved in a timely manner.

[0029] The hybridoma cells obtained in this case were sent to the China Center for Type Culture Collection for preservation, and the accession number is CCTCC NO:C202542.

[0030] (4) Mass production of monoclonal antibodies.

[0031] Healthy BALB / c mice around 10 weeks old were intraperitoneally injected with liquid paraffin, 0.5 mL per mouse. One week later, each pretreated mouse was intraperitoneally injected with 5 × 10⁻⁶ g of paraffin. 7 Hybridoma cells were collected, and ascites was extracted from mice when their abdomens became extremely distended after 10-14 days. The ascites was extracted every 2 days. The extracted ascites was centrifuged at 10,000 r / min for 10 min, and the supernatant was purified by affinity chromatography to obtain mouse anti-zebrafish IgM monoclonal antibody.

[0032] Example 3 This example demonstrates the functional validation of the mouse anti-zebrafish IgM monoclonal antibody obtained in Example 2, including the following: (1) Western blot detection.

[0033] Serum sample preparation: Wild-type zebrafish and IgM knockout zebrafish (denoted as IgM) were obtained. - / - Serum from zebrafish (IgM knocked out using CRISPR-Cas9 technology; the knockout method can be found in existing technologies and will not be described here) was diluted 10-fold with PBS, and then an equal volume of 2×Non-reducing buffer was added. The mixture was then mixed by pipetting and incubated in a 100°C metal bath for 10 min. The mixture was then placed on ice for later use or frozen at -80°C.

[0034] SDS-PAGE electrophoresis: Add an appropriate amount of 1× Tris-glycine electrophoresis buffer to the electrophoresis apparatus. Load the prepared serum sample, adding 10 μL of serum sample to each well. Turn on the power and adjust the voltage to 80 V. Once the bromophenol blue indicator reaches the separating gel, adjust the voltage to 120 V. When the bromophenol blue indicator reaches the bottom of the separating gel, stop the electrophoresis.

[0035] Transfer: Remove the SDS-PAGE gel and soak it in transfer buffer. Cut the desired size of the target protein gel block according to the size of the protein pre-stained marker, removing any unwanted parts. Take a 0.22 μm PVDF membrane, cut it to the appropriate size, soak it in methanol for 30 s, and then transfer it to the transfer buffer. Arrange the layers 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. Transfer the protein from the SDS-PAGE gel to the PVDF membrane at 300 mA semi-dry for 60 min.

[0036] Blocking: After the transfer was completed, the PVDF membrane was transferred into a PBST (0.1% Tween-20) solution containing 5% skim milk powder and incubated at room temperature for 2 h.

[0037] Primary antibody incubation: Discard the blocking solution, dilute the mouse anti-zebrafish IgM monoclonal antibody to 0.2 μg / mL with PBST solution, and incubate overnight at 4°C.

[0038] Secondary antibody incubation: Remove the primary antibody solution, wash 4 times with PBST for about 5 min each time, take HRP-labeled goat anti-mouse IgG (H+L) antibody (ABclonal) and dilute it with PBST solution at a ratio of 1:5000, and incubate at room temperature for 1 h.

[0039] ECL color development: Wash the membrane 3 times with PBST and once with PBS. Prepare an appropriate amount of ECL color development solution (purchased from Biosharp) at a 1:1 ratio. The color development solution should be prepared fresh each time. Add the color development solution to the membrane, and after full contact, place it into the imaging system. Use a chemiluminescence imaging system to analyze and take pictures.

[0040] The results of the Western blot are shown below. Figure 3 As shown, the mouse anti-zebrafish IgM monoclonal antibody provided by this invention recognizes wild-type zebrafish serum (containing IgM) but does not recognize IgM. - / - serum.

[0041] (2) Flow cytometry detection.

[0042] Zebrafish were anesthetized, and their head and kidney tissues were isolated. DMEM medium was added and the cells were ground into a cell suspension. The primary antibody was the mouse anti-zebrafish IgM monoclonal antibody provided in this invention. The cells were incubated on ice for 45 minutes, washed three times with PBS, and then incubated with FITC-labeled goat anti-mouse IgG antibody (purchased from Invitrogen) for 45 minutes. After washing three times, the cells were detected by flow cytometry.

[0043] Flow cytometry results showed that mouse anti-zebrafish IgM monoclonal antibody could separate the lymphocyte population of zebrafish head kidney into two groups, namely IgM. + and IgM - Cell population ( Figure 4 ).

[0044] (3) qPCR identification of IgM + B cells.

[0045] Further, the two cell populations in step (2) were sorted and identified using flow cytometry. IgM cells were sorted first using the same method. +B cells were sorted using a flow cytometer FACSAria™ III (BD Biosciences).

[0046] For the sorted cells, cellular RNA was extracted using a cell RNA extraction kit, as follows: ① Centrifuge the cells at 400 ×g at 4℃, then slowly aspirate and discard the supernatant with a pipette, add 500 μL LTL Lysis Buffer, and slowly pipette 3-5 times to disperse the cells; ② Add an equal volume of RNA Binding Buffer to the homogenizing solution and pipette 3-5 times; ③Place Hipure RNA mini Column I into a 2 mL collection tube, transfer all the mixture (including the precipitate) to the column, and centrifuge at 8,000 ×g for 30 s; ④ Discard the filtrate, put the column back into the collection tube, add 500 μL of Buffer RW1 to the column, let stand for 3 min, and centrifuge at 8,000 ×g for 30 s; ⑤ Discard the filtrate, put the column back into the collection tube, add 500 μL of Buffer RW2 (diluted with anhydrous ethanol) into the column, and centrifuge at 8,000 ×g for 30 s; ⑥ Discard the filtrate, put the column back into the collection tube, add 500 μL of Buffer RW2 (diluted with anhydrous ethanol) into the column, and centrifuge at 8,000 ×g for 30 s; ⑦ Discard the filtrate, put the column back into the collection tube, centrifuge the empty column at 10,000 ×g for 3 min to dry the column; ⑧ Transfer the column to a 1.5 mL RNA-free centrifuge tube, add 15-30 μL of RNase-free water to the center of the column membrane, let stand for 1 min, and centrifuge at 10,000 ×g for 1 min; ⑨ Discard the RNA column and store the RNA at -80℃.

[0047] PrimeScript with gDNA Eraser TM The RT kit reverse transcribes RNA into cDNA. The kit operates in two parts: Reverse Transcription System I (see Table 1) removes genomic DNA, and Reverse Transcription System II (see Table 2) reverses the RNA into cDNA. Table 1 Reverse transcription system I

[0048] First, prepare the reverse transcription system I according to Table 1, vortex thoroughly to mix, centrifuge, place at 42°C for 2 min on a PCR instrument, then place on ice and proceed with subsequent experimental operations as soon as possible.

[0049] Table 2 Reverse Transcription System II

[0050] Prepare reverse transcription system II according to Table 2, vortex thoroughly, centrifuge, and perform the reaction on a PCR instrument. The reverse transcription program is as follows: 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min.

[0051] The obtained cDNA template was used as a template for qPCR. The primers used are shown in Table 3, and the qPCR reaction system is shown in Table 4. The qPCR program is as follows: pre-denaturation 95℃, 3 min; denaturation 95℃, 5 s; annealing 60℃, 30 s; extension 72℃, 15 s; with 40 cycles of denaturation, annealing and extension.

[0052] Table 3. Sequences of primers used for qPCR

[0053] Table 4 qPCR reaction system

[0054] qPCR test results as follows Figure 5 As shown, IgM, IgD, and CD79a are highly expressed in IgM. + In these cells, IgZ is not expressed, and the T cell-related marker genes CD4 and CD8 are also not expressed, indicating that the monoclonal antibody provided by this invention recognizes IgM cells. + B cells.

[0055] (4) Immunofluorescence detection.

[0056] Immunofluorescence detection was performed on paraffin sections of zebrafish spleens using the following steps: ① Place the paraffin slices at 65℃ for 10 minutes; ②Dewaxing: Immerse in xylene I for 20 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min. ③ Citric acid retrieval: Preheat the hot water bath and sodium citrate system box about 1.5 hours in advance. After bathing in the water at 80-85℃ for 10 minutes, remove the sodium citrate system box and allow it to cool naturally to room temperature before taking out the sample. Wash with PBS for 3×5 min. ④ Sealing: Blot dry the liquid around the sample with absorbent paper, drop 3% BAS / PBS onto the sample, and place it in a humidified chamber at 37°C for one hour; ⑤ Primary antibody incubation: Dilute mouse anti-zebrafish IgM antibody in 3% BAS / PBS. According to the markings on the back of the sample, blot the liquid around the sample with absorbent paper, drop the diluted primary antibody onto the sample, and incubate the humidified chamber in a refrigerator at 4°C overnight. ⑥ Rinsing: Immerse the slides in PBST and rinse at the lowest speed on a shaker at room temperature. Note that the rinsing direction should be consistent with the shaking direction. Rinse with PBST for 2×10 min and with PBS for 3×10 min.

[0057] ⑦ Secondary antibody incubation: Dilute FITC-rabbit anti-mouse IgG in 3% BAS / PBS, blot dry the liquid around the sample with absorbent paper, drop the diluted secondary antibody onto the sample and place it in a humidified black box, incubate at 37°C in the dark for 40 min.

[0058] ⑧ Rinsing: Immerse the slides in PBST and rinse at the lowest speed on a shaker at room temperature. Note that the rinsing direction should be consistent with the shaking direction. PBST 2×10 min, PBS 4×10 min (change the rinsing solution each time).

[0059] ⑨ Mounting: Add 18 μL of mounting medium containing DAPI anti-quenching agent to each tablet, and slowly place it down at a 45-degree angle with tweezers to avoid air bubbles.

[0060] ⑩ Microscopic examination and image acquisition: Images were acquired using an Olympus laser confocal microscope.

[0061] The results are as follows Figure 6 As shown, the monoclonal antibody provided by this invention can recognize IgM. + B cells, and do not recognize IgM - / - B cells.

[0062] Example 4 The mouse anti-zebrafish IgM monoclonal antibody provided in Example 2 can be used to evaluate the immunization effect of the vaccine.

[0063] This study used ELISA to detect the specific antibody titer in the serum of zebrafish infected with Streptococcus agalactiae. The zebrafish used in the experiment were 3 months old and housed in the zebrafish facility of the College of Fisheries, Huazhong Agricultural University. The experimental procedure was as follows: the zebrafish were randomly divided into two groups. The control group received an intraperitoneal injection of 10 μL of sterile PBS, while the infected group received an intraperitoneal injection of 10 μL of 1×10⁻⁶ PBS. 7 cfu / mL agalactiae streptococci; after 14 days, the control group was intraperitoneally injected with 10 μL of sterile PBS, while the infected group was intraperitoneally injected with 10 μL of 1×10 7Fish were secondarily infected with cfu / mL Streptococcus agalactiae. After 14 days, blood was collected from 5 fish randomly selected from the control group and the infected group to prepare serum for ELISA detection.

[0064] The ELISA testing steps are as follows: ① Coat Streptococcus agalactiae with coating buffer (0.05 M carbonate buffer, pH 9.6), 100 μL of bacterial culture per well of the ELISA plate, and incubate overnight at 4°C; ② After washing 5 times with PBST (10 mM PBS, 0.05% Tween-20), add 250 μL of PBST solution containing 5% skim milk powder to each well and incubate at 37°C for 2 h; ③ After washing 5 times with PBST, the serum of the control group and the serum of the infected group were diluted according to the concentration gradient, and 100 μL was added to each well. PBS was used as a blank control and incubated at 37°C for 3 h. ④ After washing 5 times with PBST, dilute the mouse anti-zebrafish IgM monoclonal antibody to 1 μg / mL with PBST solution containing 5% skim milk powder, add 100 μL to each well, and incubate at 37°C for 1 h. ⑤ After washing 5 times with PBST, dilute the HRP-labeled goat anti-mouse IgG (H+L) antibody (purchased from ABclonal) with PBST solution containing 5% skim milk powder at a ratio of 1:5000, add 100 μL to each well, and incubate at 37°C for 1 h; ⑥ After washing 5 times with PBST, add 100 μL of TMB colorimetric solution to each well and develop the color at 37°C in the dark for 30 min; ⑦ Add 50 μL of stop solution (2 M H2SO4) to each well to stop the reaction for 10 min. After mixing, use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0065] ELISA test results as follows Figure 7 As shown, the monoclonal antibody provided by this invention can recognize specific IgM against Streptococcus agalactiae in serum, with a titer of 1:1000. This indicates that the antibody can be used to evaluate the immunizing effect of a vaccine.

[0066] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A hybridoma cell line secreting anti-zebrafish IgM monoclonal antibody, deposited at the China Center for Type Culture Collection on November 19, 2025, with accession number CCTCC NO:C202542.

2. A monoclonal antibody against zebrafish IgM, characterized in that, It is obtained from the hybridoma cell line described in claim 1.

3. A method for preparing the anti-zebrafish IgM monoclonal antibody as described in claim 2, characterized in that, The hybridoma cell line described in claim 1 is cultured and purified to obtain the desired product.

4. The method according to claim 3, characterized in that, The hybridoma cell line described in claim 1 was injected intraperitoneally into mice. After 10-14 days, ascites fluid was extracted, centrifuged, and the supernatant was purified.

5. The application of the anti-zebrafish IgM monoclonal antibody as described in claim 2 in the specific detection of IgM in zebrafish serum.

6. The anti-zebrafish IgM monoclonal antibody as described in claim 2, in zebrafish tissue, IgM + Applications in B cell-specific detection.

7. The application of the anti-zebrafish IgM monoclonal antibody as described in claim 2 in evaluating the immunization effect of a vaccine.

8. A reagent or kit, characterized in that, It contains the anti-zebrafish IgM monoclonal antibody as described in claim 2.

9. A method for preparing a hybridoma cell line that secretes anti-zebrafish IgM monoclonal antibody, characterized in that, The hybridoma cell line was obtained by immunizing female BALB / c mice with the fusion protein IgM-CH2-CH4 as an immunogen, and fusing the spleen cells of the immunized mice with myeloma cells; the fusion protein IgM-CH2-CH4 contains the amino acid sequence shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that, The fusion protein IgM-CH2-CH4 was prepared by expression in 293F cells.

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