Hybridoma cell, antibody and application

Abstract

The invention belongs to the technical field of biological immunology, and particularly relates to a hybridoma cell, an antibody and application. The hybridoma cell and the antibody generated by the hybridoma cell can specifically recognize siniperca chuatsi immune globulin and micropterus salmoides immune globulin, can recognize a siniperca chuatsi infectious spleen and kidney necrosis virus antibody, and develop a specific immunological tool, especially a specific antibody tool. The method is used for research on fish serological analysis, pathogen diagnosis, vaccine design and development, medicine preparation and the like, provides a new direction and method for the research, and has important significance on healthy and long-term development of fishery, breeding and ecological management.

Description

Technical Field

[0001] The invention belongs to the technical field of biological immunology, and specifically relates to a hybridoma cell line A12C3, a monoclonal antibody and an application thereof, which are of great significance for studying the humoral immune response law of mandarin fish and establishing a rapid pathogen diagnosis and fish serological analysis method with immunoglobulin as the core. Background Art

[0002] Mandarin fish (Siniperca chuatsi), also known as mandarin fish or seasonal flower fish, is an important freshwater farmed fish in China. The immune system of fish is similar to that of mammals. Immunoglobulins are important antibody molecules that play a key role in specific immune responses. Fish immunoglobulins are mainly divided into the following categories: (1) IgM: The main antibody type in fish, responsible for primary immune responses. IgM exists in tetrameric form in fish and is the most abundant antibody in serum. (2) IgD: Although less studied in mandarin fish, IgD also exists in fish and is involved in regulating B cell function. (3) IgT / IgZ: These are antibodies unique to fish and are involved in mucosal immunity. The immunoglobulin structure of mandarin fish is similar to that of other vertebrates, with heavy chains and light chains. The constant region of the heavy chain determines the class of the antibody (such as IgM), while the variable region determines the specificity of the antibody.

[0003] Monoclonal antibodies (mAbs) are antibodies produced by a single B cell clone and are highly specific to a specific antigen. Their preparation is mainly achieved through hybridoma technology, which is and Milstein in 1975. The preparation process is usually divided into the following steps:

[0004] (1) Antigen immunization: The immunoglobulin (such as IgM or IgT) of mandarin fish is used as an antigen to immunize experimental animals (such as mice, rabbits, etc.) to stimulate them to produce antibodies against mandarin fish Ig.

[0005] (2) Cell fusion: B cells are extracted from the spleen of immunized animals and fused with myeloma cells to form hybridoma cell lines. These cells can continuously produce specific antibodies and proliferate indefinitely in vitro.

[0006] (3) Screening: Hybridoma cells that can secrete specific antibodies are screened through enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay.

[0007] (4) Clonal expansion and antibody purification: The selected hybridoma cells are cultured as monoclonal clones and the antibodies are purified to obtain high-purity monoclonal antibodies.

[0008] The current status of research on fish immunoglobulin monoclonal antibodies is as follows: (1) Zebrafish IgM monoclonal antibodies: Zebrafish is a model organism, and the structure and function of its immunoglobulins have been studied in depth. Monoclonal antibodies prepared using zebrafish IgM are often used for B cell function analysis and vaccine development. [1] . (2) Atlantic salmon IgM and IgT antibody research: Monoclonal antibodies against Atlantic salmon IgM are used for immunodiagnosis of viral infections in fish. In addition, monoclonal antibodies against IgT are used to study its role in mucosal immunity. (3) Tilapia IgM monoclonal antibodies: Research on tilapia mainly focuses on vaccine efficacy evaluation, using specific IgM monoclonal antibodies to detect changes in antibodies after infection. (4) Existing mandarin fish related research: Although research on mandarin fish Ig monoclonal antibodies is relatively limited, there have been some studies on the mandarin fish immune system, which have laid the foundation for monoclonal antibody technology. For example, research on antibodies against mandarin fish viral neuronecrosis (VNN) has achieved initial results.

[0009] The research on monoclonal antibodies of mandarin fish immunoglobulin is still in its early stages, but it can draw lessons from the relevant research of other fish, such as zebrafish (Danio rerio), Atlantic salmon (Salmo salar) and tilapia (Oreochromis niloticus).

[0010] The study of mandarin fish immunoglobulins not only provides basic data for a deeper understanding of the mandarin fish immune system, but can also be used for vaccine design and development, fish disease diagnosis and prevention, and health management. For example, specific antibodies against pathogens such as bacteria and viruses can be used to detect the infection status of mandarin fish, and monoclonal antibodies can also be used as a tool for passive immunotherapy. It is of great significance to promote the healthy and long-term development of fisheries, reproduction, and ecological management. Summary of the invention

[0011] The purpose of the present invention is to provide a hybridoma cell and an antibody produced by the hybridoma cell, to prepare a specific antibody tool, to promote the immunological research of mandarin fish, to enhance the disease prevention and control capabilities and vaccine development level in aquaculture, thereby improving aquaculture efficiency, and to provide theoretical support for fish immunology.

[0012] The present invention provides a hybridoma cell, including a hybridoma cell with a deposit number of CCTCC NO: C2024121, or a subculture cell of the hybridoma cell with a deposit number of CCTCC NO: C2024121. The subculture cell of the hybridoma cell has the same biological activity as the hybridoma cell.

[0013] The hybridoma cell is prepared by fusion of a parent cell and a myeloma cell. The parent cell is a B cell obtained by immunization with the mandarin fish immunoglobulin heavy chain sequence shown in SEQ ID NO: 1 coupled with a carrier protein KLH as an antigen.

[0014] Siniperca chuatsi immunoglobulin heavy chain sequence: Cys-LVDDEEADSKYKFYTTNPVE, SEQ ID NO:1.

[0015] The myeloma cells are mouse myeloma cells, such as SP2 / 0 or NS0 mouse myeloma cells.

[0016] The subjects for immunization with the Siniperca chuatsi immunoglobulin heavy chain sequence coupled with the carrier protein KLH as an antigen include mice, rabbits, pigs, dogs, monkeys, cattle, sheep, etc. Mice include rats, mice, guinea pigs, etc., such as BALB / c mice. After immunization, the spleen of the immunized subjects is collected to extract B cells.

[0017] The method for immunizing with the Siniperca chuatsi immunoglobulin heavy chain sequence coupled to the carrier protein KLH as an antigen comprises: subcutaneous or intraperitoneal injection, the first immunization: the antigen is mixed with Freund's complete adjuvant in a ratio of 1:1 in equal volumes and then injected; 2-3 booster immunizations: two weeks after the first immunization, the antigen is mixed with Freund's incomplete adjuvant in a ratio of 1:1 and then injected, with an interval of one week between each booster immunization; sampling three days after the last immunization.

[0018] The hybridoma cells produced by the fusion of the parental cells and the myeloma cells are further screened, the steps comprising: culturing the hybridoma cells in HAT culture medium, and taking the culture supernatant for immunoassay. The immunoassay method includes immunofluorescence, enzyme-linked immunosorbent assay, immunoblotting, immunoelectrophoresis, flow cytometry and the like.

[0019] The present invention provides a method for preparing hybridoma cells, comprising the steps of: fusing parent cells with myeloma cells to prepare the hybridoma cells, wherein the parent cells are B cells obtained by immunizing with the mandarin fish immunoglobulin heavy chain sequence shown in SEQ ID NO: 1 coupled with a carrier protein KLH as an antigen.

[0020] The myeloma cells are mouse myeloma cells, such as SP2 / 0 or NS0 mouse myeloma cells.

[0021] The subjects for immunization with the Siniperca chuatsi immunoglobulin heavy chain sequence coupled with the carrier protein KLH as an antigen include mice, rabbits, pigs, dogs, monkeys, cattle, sheep, etc., and mice include rats, mice, guinea pigs, etc. After immunization, the spleen of the immunized subjects is collected to extract B cells.

[0022] The method for immunizing with the Siniperca chuatsi immunoglobulin heavy chain sequence coupled to the carrier protein KLH as an antigen comprises: subcutaneous or intraperitoneal injection, the first immunization: the antigen is mixed with Freund's complete adjuvant in a ratio of 1:1 in equal volumes and then injected; 2-3 booster immunizations: two weeks after the first immunization, the antigen is mixed with Freund's incomplete adjuvant in a ratio of 1:1 and then injected, with an interval of one week between each booster immunization; sampling three days after the last immunization.

[0023] The hybridoma cells produced by the fusion of the parental cells and the myeloma cells are further screened, the steps comprising: culturing the hybridoma cells in HAT culture medium, and taking the culture supernatant for immunoassay. The immunoassay method includes immunofluorescence, enzyme-linked immunosorbent assay, immunoblotting, immunoelectrophoresis, flow cytometry and the like.

[0024] The present invention provides an antibody and / or peptide fragment produced by a hybridoma cell with a deposit number of CCTCC NO: C2024121 or a subculture cell thereof. The antibody or peptide fragment produced by the hybridoma cell or the subculture cell thereof has the same biological activity.

[0025] The present invention provides a method for preparing an antibody or peptide fragment, comprising the steps of: culturing a hybridoma cell or a passaged cell thereof with a deposit number of CCTCC NO: C2024121, and taking a supernatant; or,

[0026] The hybridoma cells or their subcultured cells with the culture preservation number of CCTCC NO: C2024121 are placed in the abdominal cavity, and the ascites is collected.

[0027] The abdominal cavity includes the abdominal cavity of mice, rabbits, pigs, dogs, monkeys, cattle, sheep, etc. and simulated abdominal cavity or peritoneal cavity having the same or similar antibody production activity as the aforementioned abdominal cavity. Mice include rats, mice, guinea pigs, etc.

[0028] The method for preparing the antibody or peptide fragment also includes a purification operation, comprising the steps of: purifying the supernatant or ascites by chromatography. The chromatography includes affinity chromatography and ion exchange chromatography, such as Protein A / G affinity chromatography. After purification, the purity of the antibody is confirmed by SDS-PAGE, and the antibody concentration is determined by BCA method or NanoDrop.

[0029] The hybridoma cell or its progeny cell provided by the present invention, and the antibody or peptide fragment generated by the hybridoma cell or its progeny cell, are derived from the immune operation of mandarin fish immunoglobulin and are the immune products of mandarin fish immunoglobulin. The monoclonal antibody generated by the hybridoma cell line of the present invention can specifically recognize the synthesized polypeptide, mandarin fish serum and black sea bass serum, and can also specifically recognize the immunoglobulin in mandarin fish serum or black sea bass serum, especially distinguish different subtypes of immunoglobulin in serum, such as the specific recognition of mandarin fish infectious spleen and kidney necrosis virus antibody shown in the embodiment, develop immunological experimental tools, identify fish serum and serum components, and conduct fish serological analysis research.

[0030] Molecular biological techniques (such as ELISA, Western blot, etc.) utilize the binding specificity between antibodies and antigens (i.e., immunoglobulins (antibodies) are specific and can target specific antigens) to identify and distinguish different immunoglobulin subtypes through labeling and detection systems. The hybridoma cells or their progeny provided by the present invention, and antibodies or peptide fragments produced by hybridoma cells or their progeny, can specifically bind to mandarin fish immunoglobulins or black sea bass immunoglobulins, such as the antibodies specifically recognizing mandarin fish infectious spleen and kidney necrosis virus antibodies shown in the implementation mode, can develop immunological experimental tools to recognize mandarin fish immunoglobulins or black sea bass immunoglobulins, especially to recognize different subtypes of mandarin fish immunoglobulins or different subtypes of black sea bass immunoglobulins, analyze their roles in the immune response of mandarin fish or black sea bass, and study the immune response rules of mandarin fish and sea bass.

[0031] The hybridoma cells or their progeny cells provided by the present invention, and the antibodies or peptide fragments generated by the hybridoma cells or their progeny cells, can specifically recognize mandarin fish immunoglobulins or black sea bass immunoglobulins, such as the antibodies that specifically recognize mandarin fish infectious spleen and kidney necrosis virus antibodies shown in the implementation manner. Based on the principle of specific binding between antibodies and antigens, they can recognize pathogens or characteristic antigens of pathogens, and antibodies generated by immunization with pathogens or characteristic antigens of pathogens, develop immunological diagnostic tools, analyze the presence of pathogen immune antibodies or pathogen antigens in samples, detect diseases or pathogen infections, and are used for the diagnosis of diseases or pathogen infections.

[0032] Immunotherapeutic drugs usually involve monoclonal antibodies or vaccines, which recognize specific pathogens or lesion cells, enhance the body's immune response, and eliminate pathogens or lesion cells. Other drugs besides immunotherapeutic drugs can also act on pathogens or lesion cells, such as commonly used small molecule compounds, to eliminate pathogens or lesion cells and regulate the body's immune response. The hybridoma cells or their progeny provided by the present invention, and antibodies or peptide fragments produced by hybridoma cells or their progeny, specifically recognize mandarin fish immunoglobulins or black sea bass immunoglobulins, such as antibodies specifically recognizing mandarin fish infectious spleen and kidney necrosis virus antibodies shown in the implementation method, can specifically bind to antibodies generated by immune responses induced by pathogens or lesion cells, develop drug screening tools, discover promising drugs, and evaluate the therapeutic effects of preventive or therapeutic drugs for diseases or pathogen infections. Drugs include small molecule drugs and macromolecule drugs, such as antibodies, peptides, small molecule chemical drugs, etc.

[0033] After antigen immunization, immune antibodies can be produced. Molecular biological techniques (such as ELISA, flow cytometry, etc.) can be used to qualitatively or quantitatively analyze immune antibodies, thereby evaluating the immune response ability triggered by the antigen. The hybridoma cells or their progeny cells provided by the present invention, and antibodies or peptide fragments produced by hybridoma cells or their progeny cells, can specifically recognize mandarin fish immunoglobulins or black sea bass immunoglobulins, such as antibodies that specifically recognize mandarin fish infectious spleen and kidney necrosis virus antibodies shown in the embodiments, can be used to develop vaccine screening tools, evaluate the immunogenicity of antigens, and be used for vaccine design and development.

[0034] The hybridoma cells or their progeny cells provided by the present invention, and the antibodies or peptide fragments generated by the hybridoma cells or their progeny cells, can specifically recognize mandarin fish immunoglobulins or black sea bass immunoglobulins, such as the antibodies that specifically recognize mandarin fish infectious spleen and kidney necrosis virus as shown in the implementation manner. Based on the principle of specific binding between antibodies and antigens, they can recognize pathogens or characteristic antigens of pathogens, and antibodies generated by immunization with pathogens or characteristic antigens of pathogens, conduct passive immunotherapy when infection occurs, and develop immune or therapeutic drugs for the prevention or treatment of diseases.

[0035] The above-mentioned immunological experimental tools, immunological diagnostic tools, and vaccine screening tools can be in the form of reagents, kits, consumables, or instruments. The above-mentioned immunological experimental tools, immunological diagnostic tools, vaccine screening tools, and therapeutic drugs contain the hybridoma cells or their descendants provided by the present invention, and / or antibodies and / or peptide fragments produced by the hybridoma cells or their descendants.

[0036] The present invention provides an immunological experimental tool, and / or an immunological diagnostic tool, and / or a vaccine or drug screening tool, and / or an immune or therapeutic drug, which contains the hybridoma cell or its successor cell provided by the present invention, and / or antibodies and / or peptide fragments generated by the hybridoma cell or its successor cell.

[0037] As an immunological experimental tool or an immunological diagnostic tool, it is used for the identification of mandarin fish serum or black sea bass serum, and / or the identification of mandarin fish immunoglobulin or black sea bass immunoglobulin, such as the identification of mandarin fish infectious spleen and kidney necrosis virus antibodies, and is particularly suitable for the identification of different subtypes of mandarin fish immunoglobulin or different subtypes of black sea bass immunoglobulin.

[0038] The present invention provides an immunological experimental method, and / or an immunological diagnostic method, and / or a vaccine or drug screening method, comprising the steps of: using the hybridoma cell or its progeny cell provided by the present invention, and / or the antibody and / or peptide fragment generated by the hybridoma cell or its progeny cell to detect, and / or identify, and / or screen the object to be tested.

[0039] As an embodiment, the immunological experimental method is used to identify mandarin fish serum or black sea bass serum, and / or mandarin fish immunoglobulin or black sea bass immunoglobulin.

[0040] The present invention screened out an anti-siniperca chuatsi immunoglobulin cell line (A12C3) by immunizing mice and cell fusion. The monoclonal antibody produced by the cell line can specifically recognize synthetic polypeptides and siniperca chuatsi serum, and can also be used to identify largemouth bass serum. It is of great significance to study the humoral immune response of siniperca chuatsi and establish a rapid pathogen diagnosis and fish serological analysis method with immunoglobulin as the core. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Dot-blot screening of positive cell lines. NC: negative control; PC: positive control.

[0042] Figure 2 Antibody subtype identification.

[0043] Figure 3 ELISA was used to detect antibody titer.

[0044] Figure 4 Fish serum test: 1. Mandarin fish; 2. Crucian carp; 3. Largemouth bass; 4. Large yellow croaker.

[0045] Figure 5 Western blot was used to detect antibodies against infectious spleen and kidney necrosis virus in Siniperca chuatsi. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is described below in conjunction with specific embodiments.

[0047] The basic ideas for the preparation, screening and identification of the mandarin fish immunoglobulin monoclonal antibody of the present invention are as follows:

[0048] 1. Antigen preparation: In order to prepare monoclonal antibodies to mandarin fish immunoglobulins, it is first necessary to extract and purify immunoglobulins from mandarin fish as antigens. Common mandarin fish immunoglobulins include IgM and IgT. Sample source: Blood samples of mandarin fish are collected and serum is separated by centrifugation. Purification of immunoglobulins: Mandarin fish immunoglobulins are purified from serum using protein A / G affinity chromatography or column chromatography. The purification effect is verified using SDS-PAGE. Antigen confirmation: The purity and immunogenicity of immunoglobulins are verified by Western blot or ELISA experiments.

[0049] 2. Experimental animal immunization: Mice were used as experimental animals, and the mandarin fish immunoglobulin was injected into the mice to stimulate them to produce antibodies against mandarin fish Ig. Immunization dose: For the first immunization, 100 μg of purified mandarin fish Ig was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously or intraperitoneally. Immunization cycle: After the initial immunization, mandarin fish immunoglobulin and Freund's incomplete adjuvant were used for booster immunization at intervals of two weeks, usually 2 or 3 times. Final immunization: The mouse spleen was collected 3 days after the last immunization for B cell extraction.

[0050] 3. Fusion of spleen cells and myeloma cells: Spleen cell extraction: Aseptically remove the spleen of the immunized mouse and grind it to obtain a spleen cell suspension. Remove red blood cells by density gradient centrifugation to obtain single spleen cells. Myeloma cell preparation: Prepare SP2 / 0 or NS0 mouse myeloma cells, which have unlimited proliferation ability but do not secrete antibodies. Cell fusion: Mix spleen cells and myeloma cells in a ratio of 5:1, add 50% polyethylene glycol (PEG 1500) for fusion, and promote the formation of hybridoma cells.

[0051] 4. Screening of hybridoma cells: HAT medium selection: The fused cells are cultured in a medium containing HAT (hypoxanthine, methotrexate and thymidine). Unfused spleen cells die due to their short lifespan, and unfused myeloma cells cannot survive in HAT medium due to HGPRT deficiency. Only successfully fused hybridoma cells can grow. Initial screening test: 7-10 days after fusion, the supernatant of hybridoma cells that produce specific antibodies is screened using the indirect ELISA method, using mandarin fish immunoglobulin as the coating antigen.

[0052] 5. Cloning and monoclonal antibody production: Cloning: Use the limiting dilution method to separate positive hybridoma cells into single cells, ensuring that only one cell proliferates in each well to obtain a stable monoclonal cell line. Cloning verification: Use ELISA to detect the culture supernatant of each clone again to confirm the specificity of the antibody. Select clones with strong specificity for subsequent experiments.

[0053] 6. Antibody production and purification: Antibody production: The selected monoclonal hybridoma cells are cultured on a large scale. Antibodies can be secreted in the culture supernatant, or the hybridoma cells can be injected into the peritoneal cavity of mice to produce monoclonal antibodies in the form of ascites.

[0054] Antibody purification: Purify the antibody using Protein A / G affinity chromatography or ion exchange chromatography. Confirm the purity of the antibody using SDS-PAGE and determine the antibody concentration using the BCA method or NanoDrop.

[0055] 7. Antibody identification and functional verification: ELISA test: verify the binding activity of monoclonal antibody with mandarin fish Ig and calculate the antibody titer. Western blot: detect the specific binding ability of antibody to immunoglobulins such as mandarin fish IgM and IgT.

[0056] The experimental process is as follows:

[0057] 1. Preparation and screening of monoclonal antibodies against Siniperca chuatsi immunoglobulin

[0058] 1.1 Selection of Siniperca chuatsi immunoglobulin sequences

[0059] A sequence Cys-LVDDEEADSKYKFYTTNPVE (GenBank: AF327364.1; SEQ ID NO: 1) on the heavy chain of the immunoglobulin of Siniperca chuatsi was selected to synthesize a polypeptide. After the purity was ≥90%, it was coupled with a carrier protein KLH and used as an antigen to immunize BALB / c female mice.

[0060] 1.2 Antigen immunization of mice

[0061] Immunize 6-week-old BALB / c female mice with purified polypeptides, mix the purified polypeptides and adjuvants at a ratio of 1:1 to emulsify them, and repeatedly blow and aspirate to make an oil-in-water emulsion, followed by intraperitoneal injection at 50μg / mouse. For the first immunization, the purified polypeptides were mixed with Freund's complete adjuvant at a ratio of 1:1 and injected in equal volumes. The second immunization was performed two weeks after the first immunization, and the purified polypeptides and Freund's incomplete adjuvant were mixed and emulsified at a ratio of 1:1 and injected intraperitoneally. The third immunization was performed one week after the last immunization, and pure antigen was injected. One week later, the fourth immunization was performed with pure antigen. Blood was collected 3 days after the end of immunization, and the serum antibody titer was determined by indirect ELISA. Mice with high serum positive titers were selected and boosted immunization was performed three days before fusion.

[0062] 1.3 Cell fusion and culture

[0063] SP2 / 0 tumor cells were revived and cultured in advance, and immunized once intraperitoneally 3 days before cell fusion. Mice were anesthetized with ether, and spleen cells of immune mice were mixed with SP2 / 0 tumor cells at a ratio of 1:10, and then fused under the action of 50% polyethylene glycol (PEG) 4000. The fused cells were mixed with 40mL of selective culture medium containing thymocytes (HAT culture medium) and dripped on the plate, 200μL per well, dripped on 2 96-well plates, and cultured in an incubator at 37℃ with 5% CO2.

[0064] 1.4 Dot-blot screening of positive cell lines

[0065] After 10 days, observe under a microscope whether there are wells with single cell clusters and good growth. It is observed that there is a plate of cells with good growth and no contamination. All wells with single cell clusters in this plate are selected for dot blot detection. Finally, two strains of cells with good positive results are selected for subcloning, namely A12 and E4. After subcloning, 11 positive clones were identified by dot blot, and the methanol-activated PVDF membrane was transferred to the Dot blot instrument. 200 ng of polypeptide was incubated in each well, blocked with 5% skim milk for 2 hours, and 200 μL of the selected single cell well supernatant was added. Incubate at 4°C overnight, wash 3 times with PBST, add 200 μL of HRP-labeled goat anti-mouse IgG (1:3000) to each well, and incubate at 37°C for 1 hour. After washing with PBST, use ECL colorimetric solution to develop the color, and use a protein imager to take pictures and record the results. The results are as follows Figure 1 As shown, there is a cell line A12C3 with high positivity and good cell growth, which was transferred and expanded for culture.

[0066] Hybridoma cell line A12C3 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China, Postal Code: 430072) on August 22, 2024, with the deposit number CCTCC NO: C2024121, and the result was survival.

[0067] 1.5 Identification of antibody subtypes

[0068] Dilute the hybridoma cell supernatant 100 times with 1×PBST solution, then add 50μL of the sample to be tested to each sample well of the strip, and then add 1×goat anti-mouse IgM+IgG-HRP to the sample well, 50μL / well. Mix gently on a mixer, cover with a sealing film, and incubate at room temperature for 1h. Discard the liquid in the well, wash the plate 3 times with 1×PBST, and pat dry with absorbent paper. Mix the prepared color development solution (A solution: B solution = 1:100) and immediately add 100μL to each well. After coloring at room temperature in the dark for 20 minutes, add 100μL of stop solution to each well. Observe with the naked eye that the well with the darkest color or the highest OD value corresponds to the corresponding subtype ( Figure 2), the results showed that monoclonal antibody A12C3 was IgG1 type.

[0069] 2. Preparation of Monoclonal Antibodies from Ascites

[0070] Seven-week-old BALB / c female mice were kept for one week and injected intraperitoneally with 1 mL of liquid paraffin. One week later, each mouse received a single basal dose of 10 6 500 μL of hybridoma cells were added. When the mouse showed obvious abdominal swelling, the mouse ascites was extracted every 1-3 days until the mouse died. The ascites was inactivated at 56°C for 45 minutes, centrifuged at 500g for 10 minutes, and the supernatant was taken. The ascites was purified by ProteinA / G affinity chromatography, and the antibody titer was determined by indirect ELISA. Figure 3 As shown, OD(1:243K) / OD(Blank)>2.1, so the titer of the monoclonal antibody produced by the A12C3 hybridoma cell line is 1:243000.

[0071] 3. Fish serum testing

[0072] Blood was drawn from mandarin fish, crucian carp, largemouth sea bass and large yellow croaker respectively, placed in 1.5mL centrifuge tubes and left to stand at 4 degrees overnight. The next day, the blood was centrifuged at 3000rpm for 10 minutes at 4℃, and the serum was stored in a -80℃ refrigerator for later use. The methanol-activated PVDF membrane was transferred to the Dot blot instrument, and samples were spotted on the dot-blot plate in the order of mandarin fish, crucian carp, largemouth sea bass and large yellow croaker. 5μL of serum was incubated in each well, blocked with 5% skim milk for 2h, and 200μL of monoclonal antibody prepared by A12C3 hybridoma cell line (1:3000 dilution) was added, incubated at 4℃ overnight, washed 3 times with PBST, and 200μL of HRP-labeled goat anti-mouse IgG (1:3000) was added to each well, and incubated at 37℃ for 1h. After washing with PBST, the ECL colorimetric solution was used for color development, and the results were photographed and recorded with a protein imager. The results are as follows Figure 4 As shown, the monoclonal antibody produced by cell line A12C3 can recognize both mandarin fish serum and largemouth bass serum.

[0073] 4. Virus detection in mandarin fish

[0074] Western blot method was established to detect and identify antibodies against Siniperca chuatsi ISV, and the prepared Siniperca chuatsi immunoglobulin monoclonal antibody was used for identification and detection. SDS-PAGE was performed with the major capsid protein MCP of Siniperca chuatsi ISV, and the membrane was transferred to PVDF membrane by wet transfer (100V, 60min), and blocked with 5% skim milk for 2h. The serum of Siniperca chuatsi that tested positive for ISV by PCR was used as the primary antibody and incubated overnight at 4℃; the membrane was washed 5 times on a shaker at room temperature for 8min each time the next day to remove excess antibodies; Siniperca chuatsi immunoglobulin monoclonal antibody prepared by A12C3 hybridoma cell line (dilution ratio 1:1000) was added, incubated on a shaker at room temperature for 2h at 200r / min, and the supernatant was discarded, and the membrane was washed 5 times on a shaker at room temperature for 8min each time; HRP-labeled goat anti-mouse IgG was added and incubated at room temperature for 2h, and then the membrane was washed 5 times on a shaker for 8min each time. The PVDF membrane was developed for 40 seconds and observed using the Tianneng ECL gel imaging system.

[0075] It should be pointed out that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell, characterized in that The invention relates to a hybridoma cell having a deposit number of CCTCC NO: C2024121, or a subculture cell of the hybridoma cell having a deposit number of CCTCC NO: C2024121.

2. The hybridoma cell according to claim 1, characterized in that The hybridoma cells are produced by fusion of parental cells with myeloma cells.

3. The method for preparing hybridoma cells according to claim 1, characterized in that the steps include: The parental cells are fused with myeloma cells to obtain B cells obtained by immunization with the mandarin fish immunoglobulin heavy chain sequence shown in SEQ ID NO: 1 coupled with carrier protein KLH as an antigen.

4. The preparation method according to claim 3, characterized in that: The method also includes a screening step: culturing the hybridoma cells in a HAT culture medium and taking the culture supernatant for immunodetection.

5. An antibody and / or peptide fragment, characterized in that: The invention comprises antibodies and / or peptide fragments produced by a hybridoma cell with a deposit number of CCTCCNO: C2024121 or a subculture cell thereof.

6. A method for preparing an antibody or peptide fragment, characterized in that the steps include: Cultivate the hybridoma cell with the deposit number of CCTCC NO: C2024121 or its subcultured cells and collect the supernatant; or, The hybridoma cells or their subcultured cells with the culture preservation number of CCTCC NO: C2024121 are placed in the abdominal cavity, and the ascites is collected.

7. The hybridoma cell according to claim 1 or 2, and / or the antibody and / or peptide fragment according to claim 5, having at least one of the following uses: (1) Identification of mandarin fish serum or black sea bass serum; (2) Identification of mandarin fish immunoglobulin or black sea bass immunoglobulin; (3) Identification of antibodies against infectious spleen and kidney necrosis virus in mandarin fish; (3) Detection of pathogen infection; (4) evaluate the immunogenicity of the antigen; (5) Preparation of drugs for immunization or treatment; (6) Preparation of immunology experimental tools; (7) Preparation of immunological diagnostic tools; (8) Screening tools for preparing vaccines or drugs.

8. At least one of the following products prepared by a hybridoma cell according to claim 1 or 2, and / or an antibody and / or peptide fragment according to claim 5: (1) Immunology experimental tools; (2) Immunological diagnostic tools; (3) Vaccine or drug screening tools; (4) Drugs for immunization or treatment.

9. The product according to claim 8, characterized in that: The immunological experimental tool, or immunological diagnostic tool, or vaccine screening tool comprises reagents, and / or kits, and / or consumables, and / or instruments.

10. An immunological experimental method, and / or an immunological diagnostic method, and / or a vaccine or drug screening method, characterized in that: The steps include: using the hybridoma cell described in claim 1 or 2, and / or the antibody and / or peptide fragment described in claim 5 to detect, and / or identify, and / or screen the object to be detected.

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