Mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody, hybridoma cell strain and application

Abstract

The invention belongs to the field of monoclonal antibodies, and particularly relates to a mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application, a CDR region of a heavy chain variable region of the mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody comprises three sequences shown in SEQ ID NO.1-3, and a CDR region of a light chain variable region of the mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody comprises three sequences shown in SEQ ID NO.4-6; the mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody can be obtained by being secreted by a hybridoma cell strain PFM-1B7C12A1B2 with the preservation number of CCTCC (China Center for Type Culture Collection) NO: C2024234. The mouse anti-pelteobagrus fulvidraco IgM monoclonal antibody provided by the invention can be used for Western blot and ELISA specific detection of IgM in a pelteobagrus fulvidraco secretion form, and can be used for flow cytometry identification of pelteobagrus fulvidraco IgM + B cells. The monoclonal antibody is good in specificity, high in sensitivity and strong in affinity, can be used for structural analysis, immune response level detection and the like of pelteobagrus fulvidraco IgM, and lays a foundation for in-depth study of a pelteobagrus fulvidraco immune system and establishment of a vaccine immune effect evaluation method taking the antibody level as an index.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibodies, and specifically relates to a mouse anti-yellow catfish IgM monoclonal antibody, a hybridoma cell line and applications. Background Art

[0002] Yellow catfish (Pelteobagrus fulvidraco) belongs to the order Siluriformes, family Catfish, and genus Pelteobagrus. It is favored by consumers for its delicious meat, tender meat, and rich nutrition. According to the China Fisheries Statistical Yearbook, my country's yellow catfish production in 2023 was 622,600 tons, an increase of 3.81% over 2022, and it is one of my country's important characteristic freshwater farmed fish. However, with the continuous deterioration of the aquaculture water environment and the degradation of germplasm, yellow catfish diseases have occurred frequently, causing huge economic losses to the yellow catfish farming industry. Due to the continuous use of antibiotics, bacterial resistance and food health problems are becoming increasingly serious, seriously affecting the healthy and sustainable development of the aquaculture industry. Vaccination can activate the fish immune system and improve the body's resistance to specific pathogens. It will be an important means to prevent yellow catfish diseases and the mainstream direction of fish disease prevention and control in the future. However, the accurate evaluation of the immune effect of vaccines is often cumbersome, time-consuming, and lacks evaluation standards. Based on the monoclonal antibodies of fish immunoglobulins (Ig), the immune effect of the vaccine can be evaluated by analyzing the changes in the levels of specific antibodies (i.e. secretory immunoglobulins) in the vaccinated fish, which will provide a powerful tool for establishing a standard evaluation system for the immune effect of yellow catfish vaccines.

[0003] As the earliest discovered and most abundant type of immunoglobulin in bony fish, IgM has been shown to play a very important immune function in the systemic and mucosal immunity of fish. However, to date, due to the lack of yellow catfish IgM monoclonal antibodies as a tool, the study of the yellow catfish immune system has not made any progress. Therefore, the development of yellow catfish IgM monoclonal antibodies will help to gain a deeper understanding of the humoral immune response of yellow catfish, and can help establish yellow catfish antibody analysis and detection methods, and then establish a method for evaluating the immune effect of vaccines based on yellow catfish antibody levels and formulate vaccine use procedures, so as to promote the development of yellow catfish disease immune prevention and control technology with vaccination as the core. Summary of the invention

[0004] The invention provides a mouse anti-yellow catfish IgM monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell with a deposit number of CCTCC NO: C2024234.

[0005] Another object of the present invention is to provide a hybridoma cell line PFM-1B7C12A1B2, the deposit number of the hybridoma cell line is CCTCC NO: C2024234.

[0006] Another object of the present invention is to provide the use of the above monoclonal antibody or hybridoma cell line in detecting Pelteobagrus fulvidraco IgM, wherein the use includes but is not limited to preparing a Pelteobagrus fulvidraco IgM detection kit.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A hybridoma cell line PFM-1B7C12A1B2 is obtained by using Protein A affinity purified yellow catfish serum natural IgM as an antigen to immunize mice and then screened in large quantities. The hybridoma cell was deposited in the China Center for Type Culture Collection on July 16, 2024, at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: C2024234, and a classification name: Hybridoma cell line PFM-1B7C12A1B2.

[0009] The protection scope of the present invention includes:

[0010] Monoclonal antibody secreted by hybridoma cell line PFM-1B7C12A1B2.

[0011] A polynucleotide encoding the above monoclonal antibody.

[0012] The CDR region of the heavy chain variable region of the monoclonal antibody described above is characterized by comprising the following three amino acid sequences:

[0013] KSWMN (CDRH1, SEQ ID NO.1),

[0014] RIYPGDGDTNYNGNFKG (CDRH2, SEQ ID NO.2),

[0015] YNKDWYFDV(CDRH3, SEQ ID NO.3)

[0016] The CDR region of the light chain variable region is characterized by comprising the following three amino acid sequences:

[0017] KASENVVTYVS(CDRL1,SEQ ID NO.4),

[0018] GASNRYT(CDRL2, SEQ ID NO.5),

[0019] GQSYSFPLT (CDRL3, SEQ ID NO. 6).

[0020] The amino acid sequence of the heavy chain variable region of the monoclonal antibody described above is shown in SEQ ID NO.7; preferably, the polynucleotide encoding the amino acid is shown in SEQ ID NO.8;

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.9; preferably, the polynucleotide encoding the amino acid is shown in SEQ ID NO.10.

[0022] A recombinant vector comprises the above polynucleotide.

[0023] A host cell comprising the above-mentioned vector or nucleic acid.

[0024] The protection scope of the present invention also includes:

[0025] Application of the mouse anti-yellow catfish IgM monoclonal antibody, hybridoma cell line PFM-1B7C12A1B2, recombinant vector or host cell in the preparation of a yellow catfish IgM detection kit.

[0026] In the above application, preferably, the detection kit is an ELISA detection kit.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention adopts Protein A affinity purified yellow catfish serum natural IgM as antigen to immunize mice and screens a hybridoma cell strain PFM-1B7C12A1B2 that can specifically secrete mouse anti-yellow catfish IgM monoclonal antibody. The monoclonal antibody secreted by the hybridoma cell strain PFM-1B7C12A1B2 can specifically recognize the secretory form of yellow catfish IgM and membrane surface IgM. + B cells, with good specificity, high sensitivity and strong affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is the result of SDS-polyacrylamide gel electrophoresis (PAGE) in Example 1 of the present invention, detecting the purified natural IgM of yellow catfish serum under reducing conditions (Reducing) and non-reducing conditions (Non-reducing) and staining with Coomassie Brilliant Blue.

[0030] Figure 2This is a diagram showing the results of analyzing mouse anti-yellow catfish IgM monoclonal antibody subclasses by enzyme linked immunosorbent assay (ELISA) in Example 2 of the present invention.

[0031] Figure 3 This is a graph showing the results of Western blot in Example 2 of the present invention, in which the IgM band of the yellow catfish serum diluted 100 and 500 times respectively was detected using mouse anti-yellow catfish IgM monoclonal antibody under reducing conditions and non-reducing conditions.

[0032] Figure 4 The IgM in the head kidney tissue lymphocytes of yellow catfish detected by flow cytometry in Example 2 of the present invention + Results for B cells.

[0033] Figure 5 In Example 3 of the present invention, the yellow catfish serum was subjected to gel filtration chromatography column Superdex for the detection of yellow catfish serum by Western bolt and ELISA analysis of mouse anti-yellow catfish IgM monoclonal antibody. TM Results of IgM in different eluates after separation using 200Increase 10 / 300GL (GE Healthcare).

[0034] Figure 6 This is a graph showing the binding and dissociation curves of antigen proteins at different dilution concentrations and antibodies when the antibody affinity is determined using bio-layer interferometry (BLI) in Example 5 of the present invention.

[0035] Figure 7 The flow cytometry method is used to verify the effect of the supernatant of the 0th, 5th, 10th, 15th and 20th generations of the hybridoma cell line PFM-1B7C12A1B2 on the IgM in the head and kidney tissue of yellow catfish in Example 6 of the present invention. + B cell recognition results diagram.

[0036] Figure 8 This is a graph showing the results of using Western blot to verify the recognition of secretory IgM in yellow catfish serum by the supernatants of the 0th, 5th, 10th, 15th and 20th generations of hybridoma cell line PFM-1B7C12A1B2 in Example 5 of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0039] Example 1: Preparation of a mouse anti-yellow catfish IgM monoclonal antibody

[0040] This example uses HiTrap TM Protein A HP (GE Healthcare) was used to affinity purify natural IgM protein from yellow catfish serum, and it was used as an antigen to immunize mice to obtain hybridoma cell line PFM-1B7C12A1B2, which was then secreted to produce mouse anti-yellow catfish IgM monoclonal antibody. The specific process is as follows:

[0041] (1) Obtaining yellow catfish serum

[0042] Healthy yellow catfish were immersed in 30 mg / L fish anesthetic MS-222 (Sigma), and after anesthesia, they were placed in a dissecting dish, and their heads were covered with a towel. A 5 mL syringe without anticoagulant was inserted into the muscle tissue at the midline behind the anal fin until the needle reached the spine and blood entered the syringe. After the drawn blood coagulated, it was placed at 4°C overnight and centrifuged at 4°C and 3000g for 10 min the next day. The upper serum was aspirated and transferred to a clean centrifuge tube.

[0043] (2) Purification of natural IgM from yellow catfish serum

[0044] ① Equilibrate the column: Rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS).

[0045] ② Loading: Centrifuge the yellow catfish serum at 4°C and 18000g for 30 min to remove impurities, then filter through a 0.22μm filter membrane, dilute the serum with Binding buffer at a ratio of 1:3, and then filter through The sample was loaded onto the Protein A affinity purification column using pure 25L protein purification system (GE Healthcare).

[0046] ③ Neutral washing: Rinse the Protein A affinity purification column with 20 mL Binding buffer (1×PBS) to remove unbound impurities.

[0047] ④ Acid elution: Rinse 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 pre-filled with 200 μL of 1 mol / L Tris-HCl solution at pH = 9.0.

[0048] ⑤ Take the collected elution samples and perform SDS-PAGE to detect the purity of the purified yellow catfish IgM. The test results are as follows: Figure 1 As shown, 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 polymer with a size of more than 180 kDa, with a purity of 95%.

[0049] ⑥ The purified yellow catfish serum IgM was diluted with PBS at pH = 7.4 and then ultrafiltered, and finally the protein concentration was determined using a BCA kit (Biosharp).

[0050] (3) Mouse immunization

[0051] Take 5 BALB / c mice aged 6 to 8 weeks, and use the natural IgM of yellow catfish serum purified in step (2) as an immunogen to immunize the mice. The immunization route and procedure are as follows: about 50 μg of natural IgM of yellow catfish serum is fully mixed and emulsified with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points on the back of the mouse. After two weeks, three booster immunizations are performed, each with an interval of two weeks. During the booster immunization, the antigen protein is mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple points on the back. On the 7th day after the third booster immunization, tail blood is collected for ELISA to detect antibody titer.

[0052] (4) Cell fusion and culture

[0053] ① Cell fusion: Mix mouse spleen cells and mouse myeloma cells SP2 / 0 at a cell ratio of 5:1, centrifuge at 1000rpm for 10min, and discard the supernatant. Pipette 1mL PEG-4000 (Sigma) and slowly add it to the test tube while shaking; then slowly add a total of 9mL complete culture medium several times. The whole process must be carried out in a 37℃ water bath, then centrifuge at 1000rpm for 10min, remove the supernatant, and complete cell fusion.

[0054] ②Cell culture: Gently flick the cell clumps after centrifugation, resuspend them with HAT medium, and spread them in the prepared 96-well plate containing feeder cells, 200μL per well. Culture them in an incubator at 37℃ and 5% CO2. After one week, observe the clone growth under a microscope and record the number of clones in each well. When the cells grow to occupy about 1 / 3 of the bottom area of ​​the culture well, aspirate the culture supernatant for ELISA detection.

[0055] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies

[0056] ① Screening of positive clones: Coat the ELISA plate with natural IgM (2μg / mL) of yellow catfish serum, incubate overnight at 4℃, wash and block the next day, add 100μL of hybridoma cell culture supernatant to be tested to each well, incubate at 37℃ for 1h; discard the supernatant, add 100μL of HRP-labeled goat anti-mouse IgG (Proteintech) diluted at 1:5000 to each well, incubate at 37℃ for 1h; discard the supernatant, add 100μL TMB colorimetric solution (Biyuntian) to each well, color at room temperature for 10-30min, and add 50μL stop solution (2MH2SO4) to each well to terminate the reaction. Use an enzyme marker to detect the absorbance at a wavelength of 450nm, and the absorbance value is more than 2 times that of the negative control as positive.

[0057] ② Subcloning of hybridoma cells: Gently elute and count the hybridoma cells that are positive in ELISA detection from the culture plate, and continuously dilute the cell suspension with culture medium and inoculate 100 μL per well in a 96-well plate. After culturing for about 10 days, when the hybridoma cell colonies have grown to 1 / 3 of the area at the bottom of the well, begin to measure the antibody activity in the supernatant. Subclone the positive clones with antibody activity for a total of 3 times. After repeated screening, the screening conditions are: ELISA detection of high antibody titer, high antibody concentration and stable secretion in the cell supernatant, Western blot detection of the monoclonal antibody can recognize the monomer or polymer form of yellow catfish IgM, and can also be used for flow cytometry to identify yellow catfish IgM + B cells, Table 1 shows the results of ELISA and flow cytometry of different monoclonal cell lines.

[0058] Finally, a hybridoma cell line stably expressing anti-yellow catfish IgM monoclonal antibody was obtained, which was named hybridoma cell line PFM-1B7C12A1B2. It was deposited in the China Center for Type Culture Collection on July 16, 2024, with the address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024234.

[0059] Table 1 ELISA and flow cytometry results of the supernatant of the third subcloned cell line

[0060]

[0061]

[0062] ③Mass production of monoclonal antibodies: 10-week-old healthy BALB / c mice were intraperitoneally injected with liquid paraffin (0.5 mL / mouse). One week later, 5 × 10 7hybridoma cells, and after 10 to 14 days, when the abdomen of the mouse was extremely swollen, ascites was extracted, and the extraction was performed once every 2 days, and a total of 6 mL of ascites was extracted. The extracted ascites was centrifuged at 4°C and 10000 rpm / min for 10 minutes, and the supernatant was purified by Protein A affinity chromatography to obtain a total of 11 mg of purified product, namely the mouse anti-yellow catfish IgM monoclonal antibody, with a concentration of 1.83 mg / mL and a titer of 1:4096000 (as shown in Table 2).

[0063] Table 2 Results of the titer determination of mouse anti-yellow catfish IgM monoclonal antibody

[0064]

[0065]

[0066] Note: The basis for judging the antibody titer is: 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 measured at a certain dilution multiple, and N is the OD value of the negative control well), it is judged as positive. The highest dilution multiple of the antibody judged as positive is the antibody titer.

[0067] Example 2: Identification of mouse anti-Yellow Catfish IgM monoclonal antibody

[0068] (1) Detection of antibody subclasses: The process is as follows:

[0069] The purified natural IgM protein from yellow catfish serum was coated onto the ELISA plate and incubated overnight at 4°C; washed 3 times with PBST, then 100 μL of mouse anti-yellow catfish IgM monoclonal antibody was added to each well and incubated at 37°C for 2 hours; after washing 3 times, diluted HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA and IgM monoclonal antibodies were added, 100 μL per well, and incubated at 37°C for 1 hour; after washing 3 times, 100 μL of TMB colorimetric solution (Biyuntian) was added to each well and incubated at 37°C in the dark for 30 minutes; then 50 μL of stop solution (2M H2SO4) was added to each well to terminate the reaction. The absorbance was detected at a wavelength of 450 nm using an enzyme reader, and the HRP-labeled goat anti-mouse Ig subclass used in the positive reaction well was the antibody category to be tested. The identification results are as follows: Figure 2 As shown, it is shown that the subclass of the mouse anti-yellow catfish IgM monoclonal antibody prepared by the present invention is IgG2b.

[0070] (2) Western blot detection, the process is as follows:

[0071] ① Sample preparation: The serum of healthy yellow catfish was diluted 100 and 500 times with PBS, respectively, and 1 / 5 volume of 5× reduced and non-reduced SDS-PAGE loading buffer (Solarbio) was added, respectively, vortexed and mixed, heated in a 100℃ metal bath for 10 min, and placed on ice for later use or frozen at -80℃.

[0072] ②SDS-PAGE: prepare 12% and 8% Tris-glycine polyacrylamide lower separation gel and 5% upper concentration gel respectively, load the samples for electrophoresis, the electrophoresis conditions are 80V, 0.5h; 120V, 1h.

[0073] ③ Transfer: Remove the SDS-PAGE gel from the gel plate, soak it in the prepared transfer buffer (25mMTris, 192mM Glycine, 15% methanol), take a 0.22μm PVDF membrane, cut it to a suitable size, soak it in methanol for 30s to activate it, and then transfer it to the transfer buffer. Stack it from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper, ensuring that there are no bubbles in each layer, and transfer the protein on the SDS-PAGE gel to the PVDF membrane at 300mA for 60min.

[0074] ④ Blocking: After the transfer was completed, the PVDF membrane was transferred into a TBST (25 mM Tris, 150 mM NaCl, 0.1% Tween-20) solution containing 5% skim milk powder (Biosharp) and incubated at room temperature for 2 h.

[0075] ⑤ Primary antibody incubation: discard the blocking solution, dilute the mouse anti-yellow catfish IgM monoclonal antibody to 1 μg / mL with TBST solution containing 5% skim milk powder, and incubate at 4°C overnight.

[0076] ⑥ Secondary antibody incubation: remove the primary antibody solution, wash the membrane with TBST for 3 times, each time for about 10 minutes, take HRP-labeled goat anti-mouse IgG (H+L) antibody (Proteintech) and dilute it with TBST solution containing 5% skim milk powder at a ratio of 1:5000, and incubate at room temperature for 1 hour.

[0077] ⑦ECL color development: remove the secondary antibody solution, wash the membrane three times with TBST, each time for about 10 minutes, prepare an appropriate amount of ECL color development solution (Biosharp) by mixing solution A and solution B in a 1:1 ratio, add the color development solution dropwise onto the PVDF membrane, and use a chemiluminescence imager to analyze and take pictures after full contact.

[0078] (3) Flow cytometry detection, the process is as follows:

[0079] ① Isolation of total leukocytes from the head and kidney tissue of yellow catfish: Anesthetize healthy yellow catfish, cut the body cavity of the yellow catfish from the cloaca, take out the head and kidney tissue and place it in DMEM culture medium (Gibco), and cut it into 1mm squares with scissors. 3 The tissue fragments were then moved to a 100 μm cell sieve (Biosharp), and the tissue fragments were gently ground with a 2 mL syringe piston. DMEM culture medium was slowly added during the grinding process to allow single cells to pass through the 100 μm cell sieve to obtain a single cell suspension of head kidney tissue; the single cell suspension was then added to the surface of 34% and 51% discontinuous Percoll (GE Healthcare), and the white blood cells between the two layers of discontinuous Percoll were aspirated after centrifugation to obtain the total white blood cells of the head kidney tissue of yellow catfish.

[0080] ② Primary antibody incubation: Wash the total leukocytes of the head and kidney tissue of yellow catfish twice with PBS containing 2% FBS (Yeasen), then add mouse anti-yellow catfish IgM monoclonal antibody at a final concentration of 2 μg / mL, incubate on ice for 45 minutes, and gently vortex the cells once every 15 minutes.

[0081] ③ Secondary antibody incubation: Wash the cells twice with PBS containing 2% FBS, then add PE-Goat Anti-Mouse IgG (Biolegend) at a final concentration of 1 μg / mL, incubate on ice for 30 min, and gently vortex the cells once every 10 min.

[0082] ④ Flow cytometry: After washing the cells twice with PBS containing 2% FBS, resuspend the cells and filter them, and then use flow cytometry (BD) to detect IgM in the lymphocytes of the head kidney tissue of yellow catfish + The proportion of B cells.

[0083] The effect of mouse anti-Yellow Catfish IgM monoclonal antibody on secretory IgM and IgM + B cell recognition and specificity verification Figure 3-Figure 4 As shown. Under reducing conditions, the monoclonal antibody recognizes the heavy chain of yellow catfish IgM, which is about 75kDa in size. Under non-reducing conditions, the monoclonal antibody can recognize the monomer or polymer form of yellow catfish IgM. In addition, the monoclonal antibody can recognize yellow catfish IgM +B cells, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of the membrane-type IgM molecule of yellow catfish. After sequencing, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9, wherein the CDR region (CDRH1, CDRH2, CDRH3) of the heavy chain variable region respectively comprises the sequence shown in SEQ ID NO.1 to SEQ ID NO.3, and the CDR region (CDRL1, CDRL 2, CDRL3) of the light chain variable region respectively comprises the sequence shown in SEQ ID NO.4 to SEQ ID NO.6.

[0084] Example 3: Detection of serum IgM in yellow catfish using the monoclonal antibody ELISA provided by the present invention

[0085] Take 2 mL of healthy yellow catfish serum and centrifuge at 18000g for 30 min. Then filter it through a 0.22 μm filter to remove impurities. Then, filter the serum sample through PBS. pure 25L protein purification system (GE Healthcare) was loaded onto Superdex TM Gel filtration chromatography was performed on a 200Increase 10 / 300GL chromatography column (GE Healthcare), and samples with an elution volume of 6-15 mL were taken for Western Bolt and ELISA detection. The Western Bolt detection method was as described in step (2) of Example 2, and the ELISA detection method was as follows:

[0086] (1) Dilute the sample with an elution volume of 6-15 mL with coating buffer (0.05 M carbonate buffer, pH = 9.6), coat each well of the ELISA plate with 100 μL of the diluted sample, and incubate at 4°C overnight.

[0087] (2) After washing five times with PBST (10 mM PBS, 0.05% Tween-20), 250 μL of PBST solution containing 5% skim milk powder was added to each well and incubated at 37° C. for 2 h.

[0088] (3) After washing with PBST for 5 times, mouse anti-yellow catfish IgM monoclonal antibody was diluted to 1 μg / mL in PBST solution containing 5% skim milk powder as the primary antibody, 100 μL was added to each well, and incubated at 37°C for 1 h.

[0089] (4) After washing five times with PBST, HRP-labeled goat anti-mouse IgG (H+L) antibody (Proteintech) was diluted at a ratio of 1:5000 with PBST solution containing 5% skim milk powder as the secondary antibody, 100 μL was added to each well, and incubated at 37°C for 1 h.

[0090] (5) After washing five times with PBST, 100 μL of TMB colorimetric solution was added to each well and color was developed at 37°C in the dark for 30 min.

[0091] (6) Add 50 μL of stop solution (2 M H2SO4) to each well to terminate the reaction for 10 min. Mix well and measure the absorbance at 450 nm using an ELISA reader.

[0092] Test results such as Figure 5 As shown, ELISA detected OD 450 The value is consistent with the signal intensity of the band detected by Western blot, indicating that mouse anti-yellow catfish IgM monoclonal antibody can be used for ELISA to specifically detect yellow catfish natural IgM with good specificity.

[0093] In addition, the yellow catfish serum was diluted in multiples, and the ELISA experiment was performed to explore the highest dilution multiple of the yellow catfish serum that the mouse anti-yellow catfish IgM monoclonal antibody could detect. The test results are shown in Table 3, indicating that the highest dilution multiple of the yellow catfish serum that the mouse anti-yellow catfish IgM monoclonal antibody could detect was 51200 times, and the reaction was sensitive.

[0094] Table 3 ELISA verifies the highest dilution multiple of yellow catfish serum that can be detected by mouse anti-yellow catfish IgM monoclonal antibody

[0095] Yellow catfish serum dilution multiple ELISA detection of OD value 50 2.130 100 2.457 200 2.658 400 2.379 800 2.168 1600 1.936 3200 1.863 6400 1.619 12800 1.336 25600 0.898 51200 0.233 102400 0.125 PBS 0.089

[0096] Embodiment 4:

[0097] Determination of the Nucleic Acid Sequences of the Heavy and Light Chain Variable Regions of Mouse Anti-Yellow Catfish IgM Monoclonal Antibody

[0098] PFM-1B7C12A1B2 hybridoma cells in the logarithmic growth phase were taken, and total RNA was extracted using the TRizol (TAKARA) method. Oligo (dT) 20 (Invitrogen) was used as a primer for reverse transcription to generate cDNA. Then, the cDNA was used as a template and specific PCR primers were used to amplify the heavy chain variable region and light chain variable region genes respectively. The PCR products were subjected to agarose gel electrophoresis and recovered and purified, and then connected to the pMD-18T vector through TA cloning, followed by sequencing and sequence analysis, and the coding gene sequence of the heavy chain variable region was obtained as shown in SEQ ID NO.8, encoding the protein shown in SEQ ID NO.7; the coding gene sequence of the light chain variable region was shown in SEQ ID NO.10, encoding the protein shown in SEQ ID NO.9.

[0099] Embodiment 5:

[0100] Determination of the affinity of mouse anti-Yellow Catfish IgM monoclonal antibody

[0101] This example uses bio-layer interferometry (BLI) to measure the affinity of monoclonal antibodies. The specific steps are as follows:

[0102] (1) Dilute mouse anti-yellow catfish IgM monoclonal antibody to 5 μg / mL with dilution buffer (10 mM PBS + 0.02% Tween 20 + 0.2% BSA) and add to a 96-well plate, 200 μL per well;

[0103] (2) Using dilution buffer, the purified yellow catfish IgM was serially diluted at a starting concentration of 125 nM, with a total of 7 dilution gradients, and the final volume was 200 μL;

[0104] (3) The biosensor with the Protein A probe was mounted on the Gator Prime instrument and immersed in the dilution buffer for baseline measurement and recording the baseline signal to eliminate background interference;

[0105] (4) immersing the sensor into a diluted mouse anti-yellow catfish IgM monoclonal antibody solution to fix it on the sensor surface to form a biofilm layer;

[0106] (5) immersing the sensor forming the biofilm layer into the dilution buffer, recording a new baseline signal, and recording the thickness of the biofilm formed by the sensor-fixed mouse anti-yellow catfish IgM monoclonal antibody;

[0107] (6) The sensors immobilized with yellow catfish IgM monoclonal antibodies were immersed in yellow catfish IgM solutions of different dilution concentrations, respectively, and the changes in biofilm thickness caused by the binding of antigen and antibody were monitored, and the binding kinetic curves were recorded;

[0108] (7) immersing the sensor bound to the complex into a dissociation buffer for dissociation, and recording the dissociation kinetic curve;

[0109] (8) The results of the binding and dissociation curves are as follows Figure 6 As shown, the Gator Prime analysis model was used to analyze the data and calculate the affinity constant. The results showed that the affinity constant was 8.42×10 -10 M, indicating that the antibody has a strong affinity.

[0110] Embodiment 6:

[0111] Analysis on the stability of hybridoma cell lines and secreted antibodies after subculture

[0112] The hybridoma cell line PFM-1B7C12A1B2 was continuously subcultured to the 20th generation, and the cells of the 0th, 5th, 10th, 15th, and 20th generations were selected for amplification and culture, and the cell supernatant was collected. The total white blood cells of the head kidney of the yellow catfish were taken for flow cytometry verification, and the serum of the yellow catfish was taken for Western blot verification under reducing conditions. The verification results are shown in Figure 7 and Figure 8 As shown, the results showed that the cell supernatants of the 0th, 5th, 10th, 15th and 20th generations had an effect on the expression of IgM in yellow catfish. + There was no difference in the recognition of secretory IgM in serum by B cells, indicating that the hybridoma cell line had the stability of passage and the stability of secreted antibodies after passage.

[0113] In summary, the present invention successfully prepared mouse anti-yellow catfish IgM monoclonal antibody by hybridoma cell technology, which can specifically recognize yellow catfish IgM and can be used to detect yellow catfish secretory IgM and IgM + B cells are of great significance for the study of the immune system of yellow catfish and the prevention of its diseases.

[0114] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell line PFM-1B7C12A1B2, the deposit number of the hybridoma cell line is CCTCC NO: C2024234.

2. The monoclonal antibody secreted by the hybridoma cell line PFM-1B7C12A1B2 according to claim 1.

3. A polynucleotide encoding the monoclonal antibody according to claim 2.

4. The monoclonal antibody according to claim 2, wherein the sequences of CDRH1, CDRH2, and CDRH3 in the heavy chain variable region are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively, and the sequences of CDRL1, CDRL2, and CDRL3 in the light chain variable region are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

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

9. A recombinant vector comprising the polynucleotide according to claim 3.

7. A host cell comprising the polynucleotide according to claim 3 or the recombinant vector according to claim 6.

8. Use of the monoclonal antibody according to claim 2, the hybridoma cell line PFM-1B7C12A1B2 according to claim 1, the polynucleotide according to claim 3, the recombinant vector according to claim 6 or the host cell according to claim 7 in the preparation of a yellow catfish IgM detection kit.

9. The use according to claim 8, wherein the detection kit is an ELISA detection kit.

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