A high-affinity mouse anti-siniperca chuatsi IgM monoclonal antibody, hybridoma cell line and application thereof

Abstract

The present invention discloses a high-affinity mouse anti-sinipus chuatsi IgM monoclonal antibody, a hybridoma cell line and an application thereof. The mouse anti-sinipus chuatsi IgM monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the CDR region of the heavy chain variable region comprises three sequences as shown in SEQ ID NO.1~3, and the CDR region of the light chain variable region comprises three sequences as shown in SEQ ID NO.4~6; the mouse anti-sinipus chuatsi IgM monoclonal antibody can be obtained by secretion of the hybridoma cell line ScM-7H11B10C8F10 with a deposit number of CCTCC NO: C2023235. The mouse anti-sinipus chuatsi IgM monoclonal antibody provided by the present invention can be used for Western blot and ELISA to specifically detect the secretory form of IgM of the sinipus chuatsi, and can be used for flow cytometry to identify the sinipus chuatsi IgM. + B cells. This monoclonal antibody has good specificity, high sensitivity and strong affinity, and can be used for structural analysis of IgM of mandarin fish, detection of immune response levels, etc., laying a foundation for in-depth research on the immune system of mandarin fish and the establishment of a vaccine immune effect evaluation method using antibody levels as an indicator.

Description

Technical Field

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

[0002] Mandarin fish (Siniperca chuatsi), also known as Aohua fish, seasonal flower fish, and flower crucian carp, belongs to the Perciformes, Eupercidae, and Siniperca genus. It is favored by consumers for its tender, plump, thick and delicious meat and few bones. According to the China Fisheries Statistical Yearbook, the annual output of mandarin fish in my country has exceeded 300,000 tons for many consecutive years, and the aquaculture output has increased greatly in recent years. It has become one of the important freshwater aquaculture fish in my country. However, with the deterioration of the aquaculture water environment and the decline of fish resistance, mandarin fish diseases have occurred frequently, and the death of mandarin fish caused by infectious diseases such as bacterial and viral diseases is very serious, causing huge economic losses to the mandarin fish aquaculture industry. Due to the continuous use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, seriously affecting the healthy and sustainable development of aquaculture. Vaccination can activate the fish immune system and improve the body's resistance to specific pathogens. It is an important means to prevent diseases in mandarin fish and is also 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 vaccines is evaluated by analyzing the changes in antibody titers (antigen-specific Ig secreted into body fluids) in the blood and mucus of vaccinated fish. This will provide a powerful tool for establishing an evaluation standard system for the immune effect of mandarin fish vaccines.

[0003] As the earliest discovered and most abundant immunoglobulin in teleost fish, IgM has been proven to play a very important role in the systemic and mucosal immunity of fish. Therefore, the development of monoclonal antibodies against IgM in Siniperca chuatsi will help us gain a deeper understanding of the humoral immune response of Siniperca chuatsi, and help establish methods for analyzing and detecting Siniperca chuatsi antibodies, and then establish methods for evaluating the immune effects of vaccines based on the level of Siniperca chuatsi antibodies and formulate vaccine use procedures, thus promoting the development of immune prevention and control technologies for Siniperca chuatsi infectious diseases with vaccination as the core. Summary of the invention

[0004] The invention provides a high-affinity mouse anti-siniperca chuatsi IgM monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell with a deposit number of CCTCC NO: C2023235.

[0005] Another object of the present invention is to provide a hybridoma cell line ScM-7H11B10C8F10, the deposit number of the hybridoma cell line is CCTCC NO: C2023235.

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

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

[0008] A hybridoma cell line ScM-7H11B10C8F10, which was obtained by immunizing mice with Protein A affinity-purified natural IgM from mandarin fish serum after a large number of screening. The hybridoma cell line has stable passage and stable monoclonal antibody production. The hybridoma cell was deposited in the China Center for Type Culture Collection on August 1, 2023, address: Wuhan University, Wuhan, China, with a collection number of CCTCC NO: C2023235, and a classification name: Hybridoma cell line ScM-7H11B10C8F10.

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

[0010] Monoclonal antibody secreted by hybridoma cell line ScM-7H11B10C8F10.

[0011] A polynucleotide encoding the above monoclonal antibody.

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

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

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

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

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

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

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

[0019] LQYASSPPT (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; the preferred polynucleotide encoding the amino acid is shown in SEQ ID NO.9;

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.8; the preferred polynucleotide encoding the amino acid is shown in SEQ ID NO.10.

[0022] A nucleic acid molecule, which is a polynucleotide encoding the above-mentioned monoclonal antibody, or encoding the CDR region, or encoding the heavy chain and light chain variable regions;

[0023] A recombinant vector comprises the above polynucleotide.

[0024] A host cell comprising the above-mentioned vector or nucleic acid.

[0025] The protection scope of the present invention also includes:

[0026] Application of the mouse anti-siniperca chuatsi IgM monoclonal antibody, hybridoma cell line ScM-7H11B10C8F10, nucleic acid molecule, recombinant vector or host cell in the preparation of a kit for detecting siniperca chuatsi IgM.

[0027] In the above application, preferably, the detection kit is an ELISA detection kit.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses Protein A affinity purified mandarin fish serum natural IgM as an antigen to immunize mice and screens a hybridoma cell line ScM-7H11B10C8F10 that can specifically secrete mouse anti-mandarin fish IgM monoclonal antibody. Moreover, the monoclonal antibody secreted by the hybridoma cell line ScM-7H11B10C8F10 can specifically recognize the secreted form of mandarin fish IgM and IgM + B cells have good specificity, high titer, sensitive response and strong affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 in the serum of mandarin fish under reducing conditions (Reducing) and non-reducing conditions (Non-reducing) and staining with Coomassie Brilliant Blue.

[0031] Figure 2 This is a graph showing the results of analyzing mouse anti-siniperca chuatsi IgM monoclonal antibody subclasses by ELISA in Example 2 of the present invention.

[0032] Figure 3This is a graph showing the results of Western blot in Example 2 of the present invention, in which the IgM band of mandarin fish serum diluted 100 and 500 times respectively was detected using mouse anti-mandarin fish IgM monoclonal antibody under reducing conditions and non-reducing conditions.

[0033] Figure 4 The IgM in the lymphocytes of kidney tissue of Siniperca chuatsi detected by flow cytometry in Example 2 of the present invention + Results for B cells.

[0034] Figure 5 The ELISA analysis of mouse anti-siniperca chuatsi IgM monoclonal antibody in Example 3 of the present invention was performed by gel filtration chromatography column Superdex TM Graph showing the results of IgM in different elution volumes after separation using 200Increase 10 / 300GL (GE Healthcare).

[0035] Figure 6 This is a graph showing the binding and dissociation curves of antibodies at different dilution concentrations and antigen proteins when the antibody affinity is determined using surface plasmon resonance technology (SPR) in Example 4 of the present invention.

[0036] Figure 7 The flow cytometry method was used to verify the effect of the supernatant of the 0th, 5th, 10th, 15th, 20th and 25th generations of the hybridoma cell line ScM-7H11B10C8F10 on the IgM in the blood of mandarin fish in Example 5 of the present invention. + B cell recognition results diagram.

[0037] Figure 8 This is a graph showing the results of using Western blot to verify the recognition of secretory IgM in mandarin fish serum by the supernatants of the 0th, 5th, 10th, 15th, 20th and 25th generations of hybridoma cell line ScM-7H11B10C8F10 in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] 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.

[0040] Embodiment 1:

[0041] Preparation of a high-affinity mouse anti-Siniperca chuatsi IgM monoclonal antibody:

[0042] This example uses Protein A column (GE Healthcare) was used to affinity purify natural IgM protein from mandarin fish serum, and it was used as an antigen to immunize mice to obtain hybridoma cell line ScM-7H11B10C8F10, which was then secreted by the hybridoma cell line ScM-7H11B10C8F10 to produce mouse anti-mandarin fish IgM monoclonal antibody. The specific process is as follows:

[0043] (1) Obtaining mandarin fish serum

[0044] Healthy mandarin fish were immersed in 30 mg / L fish anesthetic MS-222 (Sigma). After anesthesia, they were placed in a dissecting dish and their heads were covered with a towel. A 10 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 3000 g for 10 min the next day. The upper serum was aspirated and transferred to a clean centrifuge tube.

[0045] (2) Purification of natural IgM from mandarin fish serum

[0046] ① Equilibrate the column: rinse the Protein A affinity purification column with 10 mL Binding buffer (1×PBS);

[0047] ② Loading: The mandarin fish serum was centrifuged at 4°C and 15000 rpm for 30 min to remove impurities, then filtered through a 0.22 μm filter membrane, and diluted 1:3 with Binding buffer and filtered through pure 25L protein purification system (GE Healthcare) was loaded onto a Protein A affinity purification column;

[0048] ③ Neutral washing: Wash the Protein A affinity purification column with 20 mL Binding buffer (1×PBS) to remove unbound proteins;

[0049] 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;

[0050] ⑤ Take the collected elution samples and perform SDS-PAGE to detect the purity of the purified mandarin fish IgM. The test results are as follows: Figure 1As 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 multimer with a size of more than 180 kDa, with a purity of 95%.

[0051] ⑥ The purified mandarin fish 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).

[0052] (3) Mouse immunization

[0053] Take 5 BALB / c mice aged 6 to 8 weeks, and use the natural IgM of mandarin fish 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 mandarin fish 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. Two weeks later, 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.

[0054] (4) Cell fusion and culture.

[0055] 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.

[0056] ②Cell culture: Gently flick the cell clumps after centrifugation, resuspend them with HAT medium, and spread them in the prepared 96-well plate of 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.

[0057] (5) Screening of positive clones, subcloning of hybridoma cells, and mass production of monoclonal antibodies

[0058] ① Screening of positive clones: Coat the ELISA plate with natural IgM (2μg / mL) of mandarin fish serum, incubate overnight at 4℃, wash and block the next day, add 100μL of the 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 (Biolegend) diluted at 1:2000 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 (2M H2SO4) 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 twice that of the negative control for positive results;

[0059] 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 grow 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 again, for a total of 3 times. After repeated screening, the screening conditions are: ELISA detection of high antibody titer in the cell supernatant, high antibody concentration and stable secretion, Western blot detection of the monoclonal antibody can recognize the monomer or polymer form of mandarin fish IgM, and can also be used for flow cytometry to identify mandarin fish IgM + B cells, Table 1 shows the results of ELISA and flow cytometry of different monoclonal cell lines.

[0060] Finally, a hybridoma cell line stably expressing anti-siniperca chuatsi IgM monoclonal antibody was obtained, which was named hybridoma cell line ScM-7H11B10C8F10. The hybridoma cell was deposited in the China Center for Type Culture Collection on August 1, 2023, address: Wuhan University, Wuhan, China, with the collection number CCTCC NO: C2023235, and the classification name: Hybridoma cell line ScM-7H11B10C8F10.

[0061] Table 1 ELISA and flow cytometry results of the supernatant of the third subcloned cell line

[0062] Cell line number ELISA detection of OD value <![CDATA[Flow cytometry for detecting IgM + Ratio of B cells]]> 7H11B10C8E4 3.1328 42.3％ 7H11B10C8A5 2.9752 40.9％ 7H11B10C8A7 3.1048 41.6％ 7H11B10C8D8 3.1013 42.8％ 7H11B10C8F10 3.2297 43.2％ 8G1F3A2E2 2.3569 39.1％ 8G1F3A2G6 2.6143 39.8％ 8G1F3A2D8 2.3384 26.5％ 8G1F3A2F9 2.2649 38.1％ 8G1F3A2G11 2.3058 36.9％ 8A4H3F1E3 2.7349 37.8％ 8A4H3F1C4 2.5324 29.2％ 8A4H3F1D4 2.5986 33.1％ 8A4H3F1F7 2.8898 38.6％ 8A4H3F1G9 2.5758 34.5％ 9B11H4D6E3 3.0034 39.7％ 9B11H4D6C5 2.9294 37.3％ 9B11H4D6B9 2.7881 38.1％ 9B11H4D6G9 2.8204 36.8％ 9B11H4D6B10 2.8589 28.7％

[0063] 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 6hybridoma cells, and after 10 to 14 days, when the abdomen of the mouse is extremely swollen, ascites is extracted, and the extraction is performed every 2 days, and a total of 10 mL of ascites is extracted. The extracted ascites is centrifuged at 4°C and 10000rpm / min for 10 minutes, and the supernatant is purified by affinity chromatography to obtain a total of 11.2 mg of purified product, namely the mouse anti-Siniperca chuatsi IgM monoclonal antibody, indicating that the concentration of the mouse anti-Siniperca chuatsi IgM monoclonal antibody in the ascites is 1.12 mg / mL. The initial antibody concentration is adjusted to 1 mg / mL, and the antibody titer is measured to be 1:4096000.

[0064] Table 2 Results of titer determination of a mouse anti-Siniperca chuatsi IgM monoclonal antibody

[0065]

[0066]

[0067] 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, and the highest dilution multiple of the antibody judged as positive is the antibody titer.

[0068] Embodiment 2:

[0069] Identification of Mouse Anti-Siniperca chuatsi IgM Monoclonal Antibody

[0070] (1) Detection of antibody subclasses: The process is as follows:

[0071] The purified natural IgM protein from mandarin fish serum was coated onto the ELISA plate and incubated overnight at 4°C; washed three times with PBST, then 100 μL of mouse anti-mandarin fish IgM monoclonal antibody was added to each well and incubated at 37°C for 2 hours; after washing three times, diluted HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA and IgM monoclonal antibodies were added to each well at 100 μL, and incubated at 37°C for 1 hour; after washing three 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-siniperca chuatsi IgM monoclonal antibody prepared by the present invention is IgG2a.

[0072] (2) Western blot detection, the process is as follows:

[0073] ① Sample preparation: After the serum of healthy mandarin fish was diluted 100 and 500 times with PBS, 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℃;

[0074] 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.

[0075] 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.

[0076] 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.

[0077] ⑤ Primary antibody incubation: discard the blocking solution, dilute the mouse anti-siniperca chuatsi IgM monoclonal antibody to 1 μg / mL with TBST solution containing 5% skim milk powder, and incubate at 4°C overnight.

[0078] 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 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.

[0079] 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.

[0080] (3) Flow cytometry detection, the process is as follows:

[0081] ① Isolation of total leukocytes from the head and kidney tissues of mandarin fish: Anesthetize healthy mandarin fish, cut the body cavity of mandarin fish from the cloaca, take out the head and kidney tissues, place them in DMEM culture medium, and cut them into 1 mm pieces with scissors. 3The tissue fragments were then transferred to a 100 μm cell sieve, and the tissue fragments were gently pressed with a 2 mL syringe piston. During the pressing process, DMEM culture medium was slowly added 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 liquid surface of 34% and 51% discontinuous Percoll (GE Healthcare), and the white blood cells between the two layers of discontinuous Percoll were aspirated after gradient centrifugation to obtain the total white blood cells of the head kidney tissue of Siniperca chuatsi;

[0082] ② Primary antibody incubation: Wash the total leukocytes of the kidney tissue of mandarin fish twice with PBS containing 2% FBS (Yeasen), then add mouse anti-mandarin fish 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;

[0083] ③ Secondary antibody incubation: Wash the cells twice with PBS containing 2% FBS, then add APC 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;

[0084] 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 lymphocytes of kidney tissue of Siniperca chuatsi + The proportion of B cells.

[0085] The effect of mouse anti-Siniperca chuatsi IgM monoclonal antibody on the secretory form of Siniperca chuatsi IgM and IgM + B cell recognition and specificity verification Figure 3-Figure 5 As shown. Under reducing conditions, the monoclonal antibody recognizes the heavy chain of the secretory form of IgM of Siniperca chuatsi, which is about 75kDa in size. Under non-reducing conditions, the monoclonal antibody can recognize the monomer or polymer form of the secretory form of IgM of Siniperca chuatsi. In addition, the monoclonal antibody can recognize Siniperca chuatsi IgM + B cells, indicating that the monoclonal antibody can recognize the antigenic epitope on the surface of the mandarin fish membrane type IgM molecule. 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.8, wherein the CDR region of the heavy chain variable region comprises the sequence shown in SEQ ID NO.1 to SEQ ID NO.3, and the CDR region of the light chain variable region comprises the sequence shown in SEQ ID NO.4 to SEQ ID NO.6.

[0086] Embodiment 3:

[0087] Detection of IgM in Siniperca chuatsi serum by using the monoclonal antibody ELISA provided by the present invention

[0088] Take healthy mandarin fish serum and pass it through molecular sieves, and take samples with elution positions of 6-15mL for ELISA detection. The detection steps are as follows:

[0089] (1) Dilute the sample with elution position 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;

[0090] (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;

[0091] (4) After washing with PBST for 5 times, mouse anti-siniperca chuatsi 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;

[0092] (5) After washing with PBST five times, HRP-labeled goat anti-mouse IgG (H+L) (Proteintech) antibody was diluted at a ratio of 1:2000 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;

[0093] (6) After washing with PBST five times, 100 μL of TMB colorimetric solution was added to each well and color was developed at 37°C in the dark for 30 min;

[0094] (7) 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.

[0095] Test results such as Figure 7 As shown, mouse anti-Siniperca chuatsi IgM monoclonal antibody can be used for ELISA to specifically detect Siniperca chuatsi natural IgM with good specificity.

[0096] In addition, the mandarin fish serum was diluted in multiples, and the highest dilution multiple of mandarin fish serum that could be detected by the mouse anti-mandarin fish IgM monoclonal antibody was explored by ELISA experiment. The test results are shown in Table 3, indicating that the highest dilution multiple of mandarin fish serum that could be detected by the mandarin fish IgM monoclonal antibody was 51200 times.

[0097] Table 3 ELISA verification of the highest dilution multiple of Siniperca chuatsi serum detected by mouse anti-Siniperca chuatsi IgM monoclonal antibody

[0098] Dilution multiple of mandarin fish serum ELISA detection of OD value 50 2.611 100 2.865 200 2.813 400 2.761 800 2.453 1600 2.081 3200 1.798 6400 1.612 12800 1.103 25600 0.702 51200 0.439 102400 0.137 PBS 0.088

[0099] Embodiment 4:

[0100] Monoclonal antibody affinity determination

[0101] This example uses surface plasmon resonance technology (SPR) to measure antibody affinity. The specific steps are as follows:

[0102] (1) Install the COOH chip according to the standard operating procedure of the OpenSPRTM instrument.

[0103] (2) Start running at maximum flow rate (150 μL / min) with PBST as the detection buffer.

[0104] (3) After reaching the signal baseline, load 200 μL of IPA (isopropanol) and run for 10 s to eliminate bubbles. After reaching the baseline, rinse the sample loop with buffer and evacuate with air.

[0105] (4) After the signal reaches the baseline, adjust the PBST flow rate to 20 μL / min.

[0106] (5) Load EDC / NHS (1:1) solution to activate the chip.

[0107] (6) Load 200 μL of Protein A affinity-purified mandarin fish IgM diluted in PBST and run for 4 min. Rinse the sample loop and empty it with air.

[0108] (7) Load 200 μL of Blocking solution, rinse the sample loop with PBST, and evacuate it with air. Observe the baseline for 5 minutes to ensure stability.

[0109] (8) The antibody to be detected was diluted with PBS to 80 nm, 40 nm, 20 nm, 10 nm, 5 nm and 2.5 nm, respectively, and loaded at 20 μL / min. The binding time of antigen and antibody was 4 min; the natural dissociation time was 6 min.

[0110] (9) The results of the binding and dissociation curves are as follows Figure 6 As shown, the affinity constant analysis was performed using the One To One analysis model and TraceDrawer (Ridgeview Instruments ab, Sweden) software. The results showed that the affinity constant was 6.29×10 -9 M, indicating that the antibody has a strong affinity.

[0111] Embodiment 5:

[0112] Analysis on the stability of hybridoma cell lines and secreted antibodies after subculture

[0113] The hybridoma cell line ScM-7H11B10C8F10 was continuously subcultured to the 25th generation, and the cells of the 0th, 5th, 10th, 15th, 20th and 25th generations were selected for amplification and culture, and the cell supernatant was collected. The total leukocytes of the blood of mandarin fish were verified by flow cytometry, and the serum of mandarin fish was verified by Western blot under reducing conditions. The verification results are shown in Figure 7 and Figure 8 As shown, the results showed that the cell supernatants of passages 0, 5, 10, 15, 20 and 25 were positive for Siniperca chuatsi IgM + 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.

[0114] In summary, the present invention successfully prepared mouse anti-Siniperca chuatsi IgM monoclonal antibody by hybridoma cell technology, which can specifically recognize Siniperca chuatsi IgM and can be used to detect Siniperca chuatsi secretory IgM and IgM + B cells are of great significance for the study of the immune system of mandarin fish and the prevention of infectious diseases.

[0115] 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 ScM-7H11B10C8F10, the deposit number of the hybridoma cell line is CCTCC NO: C2023235.

2. The monoclonal antibody secreted by the hybridoma cell line ScM-7H11B10C8F10 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, CDRL 2, and CDRL 3 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.

8. A recombinant vector comprising the nucleic acid molecule according to claim 3.

7. A host cell comprising the nucleic acid molecule according to claim 3 or the recombinant vector according to claim 6.

8. Use of the IgM monoclonal antibody according to claim 2, the hybridoma cell line ScM-7H11B10C8F10 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 kit for detecting IgM of mandarin fish.

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

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