Monoclonal antibody specifically binding to clostridium perfringens alpha toxin recombinant protein, hybridoma cell strain and application thereof

Abstract

The application belongs to the technical field of bioengineering, and discloses a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. α The application provides a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. α The application provides a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. α The application provides a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. α The application provides a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. α The application provides a monoclonal antibody of a recombinant protein of Clostridium perfringens toxin, a hybridoma cell strain and application. The kit uses a mouse monoclonal antibody of a recombinant protein of Clostridium perfringens toxin as a competitive antibody, can detect serum antibodies from various animals, avoids the limitation of species of sample sources, has good repeatability, has relatively loose technical requirements in operation, can be widely used in production, and is applied to epidemiological investigation and vaccine immune level monitoring of Clostridium perfringens.

Description

Technical Field

[0001] This invention relates to a specific binding of Clostridium putrefactive bacteria. α Monoclonal antibodies, hybridoma cell lines, and applications of recombinant toxin proteins belong to the field of biotechnology. Background Technology

[0002] Clostridium putrefactivee ( Clostridium septicum *Gastroparesis* is a Gram-positive anaerobic bacterium, the first pathogenic anaerobic bacterium isolated from humans. It produces spores and is one of the main pathogens of gas gangrene in humans and animals. This bacterium is commonly found in nature in spore form. When the host's internal environment changes, it can cause gas gangrene in humans and malignant edema in various animals such as pigs, horses, cattle, sheep, chickens, and deer. When this pathogen infects sheep via the digestive tract, it can cause a serious infectious disease—sheep enterocolitis (EAC). Braxy This disease has a rapid onset, spreads quickly, and has a short course, rapidly causing death in animals such as sheep within a short period, posing a significant threat to livestock farming. The pathogenic factor of this bacterium is... alpha, beta, gamma and delta Exotoxins. Among them, α The toxin is the main lethal virulence factor, and also the main immune protective antigen.

[0003] Clostridium putrefactivee α The toxin, encoded by the csa gene, is a pore-forming cytolytic agent with hemolytic, necrotizing, and lethal properties, and is an essential virulence factor for *Clostridium putrefactiveans*. The toxin has an isoelectric point (pI) of 8.4, is not heat-resistant, and is easily inactivated by heat. Once *Clostridium putrefactiveans* enters the organism... α The toxin is secreted as an inactive 46 kDa monomeric form that can bind to a glycosylphosphatidylinositol (GPI)-anchored protein via a tryptophan-rich sequence located in the C-terminal region on a lipid raft. GPI receptors are expressed on the surface of many cells, therefore... α The toxin has broad host cell specificity. α After the toxin binds to the GPI receptor, the inactive monomer is cleaved by proteases within the host cell, increasing the sites for protein-protein interactions and transforming it into its active form. α The toxin polymerizes to form oligomers with a molecular weight of approximately 230 kDa, exhibiting concentration dependence and tolerating temperature and SDS without depolymerization. After oligomerization, α The toxin can form heptameric ion exchange pores on the surface of host cells. These pores facilitate the unregulated entry of extracellular ions (including calcium) into the poisoned cells, ultimately leading to cell lysis and death. In the digestive tract, the toxin can cause inflammation and necrosis of the digestive tract mucosa. The toxin enters the body via the bloodstream, stimulating the central nervous system and causing acute shock and rapid death in sheep. Therefore, in the development of next-generation Clostridium perfringens genetically engineered vaccines, Clostridium putrefactiveum is crucial.α The role of toxin proteins is also important. Vaccination is the most effective means of preventing and controlling Clostridium perfringens infection. Serological detection methods are used to evaluate the levels of Clostridium perfringens in the serum of vaccinated animals. α Antibody levels of the toxin protein are crucial for testing the quality of Clostridium putrefactive bacteria vaccines and for developing high-quality vaccines; however, there are currently no commercially available Clostridium putrefactive bacteria vaccines in China. α Toxin antibody detection ELISA kit.

[0004] Monoclonal antibody technology involves fusing antibody-producing single B lymphocytes with bone marrow tumor cells to obtain hybrid cells that can both produce antibodies and proliferate indefinitely. These hybrid cells are then used to produce highly specific monoclonal antibodies. Monoclonal antibodies can be used in the clinical treatment of diseases and to establish various ELISA detection methods. Competitive ELISA is a method for qualitative or quantitative detection of antigens or antibodies. Its principle is based on the competitive binding of antigens and antibodies, therefore requiring relatively low sample purity and allowing detection even in complex sample matrices. Competitive ELISA is simple to operate, low in cost, and suitable for detecting large quantities of serum samples from various animals, making it suitable for large-scale screening. In previous studies, our laboratory established a detection method for Clostridium putrefactive bacteria in sheep serum. α Indirect ELISA methods using toxin antibodies can be used to identify putrefactive Clostridium perfringens in serum. α The level of toxin antibodies is important. However, indirect ELISA has a relatively high false positive rate and only detects antibody levels in the serum of specific animals. Therefore, it is necessary to establish a detection system that can detect Clostridium putrefactive bacteria in the serum of multiple animals. α Methods for detecting toxin antibody levels. Preparation of *Clostridium putrefactiveii*. α A monoclonal antibody against the toxin protein was developed, and this monoclonal antibody was used to develop a drug capable of targeting Clostridium putrefactive bacteria. α Competitive ELISA kits for toxin antibody detection will have significant application value in the prevention and control of Clostridium putrefactive disease. Summary of the Invention

[0005] The purpose of this invention is to provide a method capable of secreting substances that specifically bind to Clostridium putrefactive bacteria. α Hybridoma cell line Clostridium putrefactivee containing monoclonal antibodies against recombinant toxin proteins α Toxin -8H2.

[0006] Another object of the present invention is to provide a specific binding to Clostridium putrefactive bacteria. α Monoclonal antibodies against recombinant toxin proteins.

[0007] Another objective of this invention is to provide a method for detecting putrefactive Clostridium perfringens in serum. α Competitive ELISA kit for toxin antibodies.

[0008] In order to achieve the above object, the technical scheme adopted by the present application is:

[0009] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein. α The present application provides a hybridoma cell strain of the monoclonal antibody of the Clostridium perfringens toxin recombinant protein. α The present application provides a hybridoma cell strain of the monoclonal antibody of the Clostridium perfringens toxin recombinant protein. α The present application provides a hybridoma cell strain of the monoclonal antibody of the Clostridium perfringens toxin recombinant protein.

[0010] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein. α The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein.

[0011] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein.

[0012] EVKLKESGAELVRPGTSVKLSCKASGYKFSSYWIHWVKQRPGQGLEWIGLIDPSDSYSNNNQKFKGKATLTVDTSASTAYIHLSRLTSEDSAVYYCAKGPHYSAMDYWGQGTSVTVSS

[0013] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein.

[0014] DIQMTQSPASLSTSVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNARTLAEGLPSRFSGSGSGTQFSLKISSLQPEDFGSYYCQHHYGAFRTFGGGTKLEIKR

[0015] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein.

[0016] The present application provides a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein.

[0017] GAGGTGAAGCTGAAGGAGTCTGGGGCTGAGTTGGTGAGGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACAAGTTCAGTAGCTACTGGATTCACTGGGTAAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGACTGATCGATCCTTCTGATAGTTACAGTAATAACAATCAAAAGTTTAAGGGCAAGGCCACATTGACTGTAGACACATCCGCCAGCACAGCCTACATACACCTCAGCCGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAAGGGCCCCCATTACTCTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0018] Light chain variable region nucleotide sequence (SEQ ID NO. 4):

[0019] GATATCCAGATGACTCAGTCTCCAGCCTCCCTATCTACATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAACATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAGAACCTTAGCAGAAGGTTTGCCATCAAGGTTCAGTGGCAGTGGATCAGGTACACAGTTTTCTCTGAAGATCAGCAGCCTGCAGCCTGAGGATTTTGGGAGTTATTACTGTCAACATCATTATGGTGCTTTCCGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAACGG

[0020] Specifically binds Clostridium perfringens α The monoclonal antibody to the recombinant protein of Clostridium perfringens toxin is produced by the hybridoma cell strain with the preservation number CCTCC NO: C202535. The monoclonal antibody specifically binds to the recombinant protein of Clostridium perfringens toxin and competes with the positive serum of Clostridium perfringens for binding to Clostridium perfringens α Specifically binds Clostridium perfringens α The monoclonal antibody to the recombinant protein of Clostridium perfringens toxin is produced by the hybridoma cell strain with the preservation number CCTCC NO: C202535. The monoclonal antibody specifically binds to the recombinant protein of Clostridium perfringens toxin and competes with the positive serum of Clostridium perfringens for binding to Clostridium perfringens αThe monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification.

[0021] The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification.

[0022] The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification.

[0023] The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification. α The monoclonal antibody of the toxin recombinant protein is prepared by the following method: the hybridoma cell strain CCTCC NO: C202535 is injected into the abdominal cavity of BALB / c female mice, the ascites of the BALB / c female mice is obtained, the light yellow ascites in the middle layer is collected after centrifugation, and the monoclonal antibody of the toxin recombinant protein is obtained after purification.

[0026] (1) The present application uses soluble expression Clostridium perfringens α Toxin protein as antigen, immunizes mice, takes mouse spleen cells and myeloma SP2 / 0 cells fusion, and a hybridoma cell strain Clostridium perfringens αToxin-8H2, the preservation number of which is CCTCC NO: C202535. The CCTCC NO: C202535 cell strain of the present application is inoculated into the abdominal cavity of a mouse, and the purified antibody is obtained by collecting the mouse ascites and purifying the antibody, so that the purified monoclonal antibody specifically combined with Clostridium perfringens toxin is obtained α The monoclonal antibody specifically combined with the toxin ensures the specificity of the reaction.

[0027] (2) The kit provided by the present application uses Clostridium perfringens toxin as an antigen, and the specific monoclonal antibody prepared by using the antigen as a competitive antibody, so that the Clostridium perfringens toxin protein competitive ELISA antibody detection kit is prepared. α α The Clostridium perfringens toxin protein competitive ELISA antibody detection kit has higher specificity and sensitivity than the indirect ELISA kit currently used for detecting Clostridium perfringens serum antibodies.

[0028] (3) The kit provided by the present application uses the monoclonal antibody of the Clostridium perfringens toxin recombinant protein as a competitive antibody, so that serum antibodies of various animal origins can be detected, and the species limitation of sample sources is avoided. There is no related product at home and abroad at present, and the technical gap at home and abroad is filled, and a technical problem urgently needed to be solved in the production of the breeding industry is solved α

[0029] (4) The Clostridium perfringens toxin protein competitive ELISA antibody detection kit developed into a commercial product can evaluate the immunization effect of the Clostridium perfringens triple quadruple vaccine currently used in the sheep breeding industry, and can also conduct a serological investigation on sheep that have not been immunized with the Clostridium perfringens vaccine. α

[0030] (5) The technical scheme of the present application is a competitive ELISA technology, and the mouse-derived monoclonal antibody is used as a competitive antibody, so that the antibody is easy to prepare and produce in a large amount. The technology can realize high-throughput detection, and related professionals can operate according to the specification, and has universality. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a preparation method flowchart of the monoclonal antibody of the present application specifically combined with the Clostridium perfringens toxin recombinant protein. α

[0032] Figure 2 is a schematic diagram of the Clostridium perfringens toxin recombinant protein of the present application; M: protein Marker relative molecular mass standard; 1, 2: pGEX-4T-1 bacteria supernatant, precipitation; 3, 4: uninduced bacteria supernatant, precipitation; 5, 6: induced bacteria supernatant, precipitation; α

[0033] Figure 3 is a recombinant Clostridium perfringens toxin protein of the present application; M: protein Marker relative molecular mass standard; 1, 2: pGEX-4T-1 bacteria supernatant, precipitation; 3, 4: uninduced bacteria supernatant, precipitation; 5, 6: induced bacteria supernatant, precipitation; α ​​​​​Purification of toxin protein; M: PM2510 relative molecular mass standard; M: PM2510 relative molecular mass standard; 1: flow-through liquid; 2: 100 mM imidazole; 3: 150 mM imidazole; 4-5: 200 mM imidazole; 6-7: 250 mM imidazole; 8-9: 300 mM imidazole. Low concentration of imidazole (100 mM, 150 mM imidazole) removes impurities, and high concentration of imidazole (200 mM, 250 mM, 300 mM imidazole) elutes the target protein.

[0034] Figure 4 Clostridium perfringens toxin of the present application α Purification of recombinant toxin protein monoclonal antibody; M: Protein Marker relative molecular mass standard; 1: hybridoma cell; 2: purified Clostridium perfringens toxin α Recombinant toxin protein monoclonal antibody. The purified ascites showed a single band at 55 kDa (heavy chain) and 25 kDa (light chain), and the ascites was successfully prepared and purified. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0036] Example 1

[0037] 1. Clostridium perfringens toxin α Preparation of recombinant toxin protein

[0038] According to the Clostridium perfringens alpha toxin gene sequence published by UniProt protein database, the codons were optimized and sent to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis and ligation into pGEX-4T-1 vector.

[0039] The recombinant plasmid was transformed into competent Escherichia coli BL21 (DE3) pLysS, and after PCR amplification and sequencing identification, the recombinant expression strain E. coli BL21 (pGEX-4T-1- α ) was obtained. E. coli BL21 (pGEX-4T-1- α ) strain was cultured at 37°C, 200 r / min to OD 600 value of 0.6~0.8, induced with 0.5 mM IPTG for 13 h. The bacterial solution after induction was centrifuged, the precipitate was washed with pH 7.4 PBS, resuspended, then the bacterial body was broken by ultrasonic wave, centrifuged, and the supernatant was taken to obtain the soluble expression of Clostridium perfringens αToxin recombinant protein, purified by GST-tag protein purification kit, to obtain purified Clostridium perfringens α Toxin recombinant protein; the purity is more than 90% through gray scale analysis.

[0040] Figure 2 Clostridium perfringens α Toxin recombinant protein; the target protein is expressed in a soluble form at about 60 kDa.

[0041] Figure 3 Clostridium perfringens α Toxin protein purification diagram; low concentration of imidazole (100 mM, 150 mM imidazole) removes impurities, and high concentration of imidazole (200 mM, 250 mM, 300 mM imidazole) elutes the target protein.

[0042] Figure 4 Clostridium perfringens α Toxin recombinant protein monoclonal antibody purification diagram; the ascites after purification appears a target band at 55 kDa (heavy chain) and 25 kDa (light chain) and the band is single, and the ascites is successfully prepared and purified.

[0043] 2, secreting specific binding Clostridium perfringens α Screening of hybridoma cell strains of monoclonal antibodies of toxin recombinant protein of Clostridium perfringens

[0044] Select 6-8 week old BALB / c female mice 6, mix Clostridium perfringens alpha toxin recombinant protein and Freund's complete adjuvant 1:1 to perform the first mouse immunization; subsequent immunization is performed every 2 weeks, and the adjuvant used is Freund's incomplete adjuvant, and the volume ratio of Clostridium perfringens α Toxin recombinant protein and Freund's incomplete adjuvant; a total of 4 times of immunization, and the antigen dose is 50 μg per mouse per time. Two weeks after the fourth immunization, the serum of the mouse is collected, and Clostridium perfringens α Toxin recombinant protein as antigen, indirect ELISA is used to detect the titer of Clostridium perfringens alpha toxin protein antibody in mouse serum, and P / N≥2.0 is determined as positive, and the titer reaches more than 1:50000 to be immune qualified. Take the spleen cells of the immune qualified mouse, and fuse with SP2 / 0 myeloma cells, and the fusion ratio is 5:1. The cells after fusion are semi-replaced and fully replaced on the 7th day and the 10th day after fusion, respectively. On the third day after full replacement, hybridoma cells secreting anti-Clostridium perfringens α Toxin protein antibody are screened by indirect ELISA method. The positive wells screened in the first screening are fully replaced for the second screening. The wells still positive in the second screening are expanded and frozen, and 3-5 times of subcloning are continuously performed until the positive rate is 100%, and the positive cell strain is frozen in a liquid nitrogen tank.

[0045] BALB / c mice were sensitized by intraperitoneal injection of 1 mL of autoclaved liquid paraffin, followed by intraperitoneal injection of 1.0 × 10⁻⁶ mol / L paraffin 7 days later. 6 A number of well-growing positive hybridoma cells were collected. The peritoneal cavity of mice was observed daily, and ascites fluid was collected when the mice became distended to the point of difficulty in movement. The ascites fluid obtained in this invention was purified using a Thermo Protein G purification column, and its purity was observed by SDS-PAGE. The concentration was also determined, and the fluid was stored at -80°C for later use.

[0046] This invention specifically binds to putrefactive Clostridium perfringens. α Hybridoma cell line Clostridium putrefactivee containing monoclonal antibodies against recombinant toxin proteins α Toxin-8H2 was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; accession number CCTCC NO: C202535; scientifically described as the hybridoma cell line *Clostridium putrefactivee*. α Toxin-8H2Hybridoma cell line putrefactive Clostridium perfringens α Toxin-8H2; This culture was received by this depository on January 2, 2025, and has been registered.

[0047] 3. Specific binding to putrefactive Clostridium perfringens α Preparation method of monoclonal antibody against recombinant toxin protein

[0048] Secretion-specific binding of putrefactive Clostridium α Hybridoma cell lines secreting specific binding Clostridium putrefactive bacteria containing recombinant toxin proteins α Monoclonal antibodies against recombinant toxin proteins. Specifically bind to *Clostridium putrefactiveii*. α Monoclonal antibodies against recombinant toxin proteins and Clostridium putrefactive bacteria α The toxin protein specifically binds to and interacts with Clostridium putrefactive bacteria. α Toxin-positive serum competitively binds to Clostridium putrefactive bacteria α Toxin protein.

[0049] like Figure 1 As shown, it specifically binds to Clostridium putrefactive bacteria. α Methods for preparing monoclonal antibodies against recombinant toxin proteins include:

[0050] S101, the hybridoma cell line CCTCC NO:C202535 was inoculated;

[0051] S102, obtain ascites fluid from female BALB / c mice, centrifuge, and collect the middle layer of pale yellow ascites fluid;

[0052] S103, obtained after purifying ascites fluid, contains putrefactive Clostridium. α Monoclonal antibodies that specifically bind to recombinant toxin proteins.

[0053] Clostridium perfringens α Establishment of toxin competitive ELISA antibody detection method

[0054] Clostridium perfringens α The optimal coating concentration of the toxin recombinant protein is 3.0 μg / mL, 100 μL / well; the serum is 1:1, 50 μL / well; the dilution multiple of the monoclonal antibody (initial concentration 1 mg / ml) is 1:2000, 50 μL / well, the incubation time is 90 min; the dilution multiple of the HRP-labeled goat anti-mouse antibody is 1:15000, 100 μL / well, the incubation time is 30 min. The substrate solution TMB is 100 μL / well, the incubation time is 15 min; the termination solution is 50 μL / well, and the OD450 value is detected immediately after addition.

[0055] The coating procedure of the antigen is as follows: the antigen stored at-75℃ is thawed, diluted to 3.0 μg / mL with a pH 7.2 phosphate buffer, added to an enzyme-labeled plate, 100 μL / well, and transferred to 4℃ for coating for 16 h. The next day, the antigen solution is shaken off, blocked with 1% BSA after washing, 100 μL / well, incubated at 37℃ for 90 min, and then washed. Then, a protein stabilizer is added, 100 μL / well, incubated at room temperature for 10 min, shaken dry, and blown for 1 h in a clean bench. Finally, it is placed in a packaging bag and vacuumed.

[0056] The preparation method of the standard positive serum in the kit is as follows: a 6-month-old sheep is selected, and the sheep is immunized with Clostridium perfringens α The toxin protein is mixed with Freund's complete adjuvant at a ratio of 1:1 for the first immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. α The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. α The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. α The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. β 1 The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. β 2 The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. epsilon The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time. α The toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the second immunization; after an interval of 2 weeks, the toxin protein is mixed with Freund's incomplete adjuvant at a ratio of 1:1 for the third immunization; and the antigen dose is 500 μg / sheep / time.

[0057] HRP labeled goat anti-mouse antibody in the detection kit of the application is purchased from Abeam Company; monoclonal antibody diluent, enzyme-labeled antibody diluent, TMB developing solution, stop solution, 50-fold concentrated washing solution are all purchased from Lanzhou Animal Research Biotechnology Co., Ltd. of Chinese Academy of Agricultural Sciences.

[0058] Clostridium perfringens in Example 3 α Toxin competitive ELISA antibody detection kit

[0059] Establishment of detection kit: after various reagents are complete, assemble into detection Clostridium perfringens α Toxin serum antibody competitive ELISA kit. It is suggested that the kit includes: coating Clostridium perfringens α Toxin recombinant antigen enzyme-labeled plate 2 pieces (96 wells / piece); 25-fold PBST concentrated solution 1 bottle (60 mL / bottle); standard positive serum 1 tube (0.6 mL / tube); standard negative serum 1 tube (0.6 mL / tube); monoclonal antibody working solution 1 bottle (12 mL / bottle); HRP labeled goat anti-mouse secondary antibody working solution 1 bottle (25 mL / bottle); TMB substrate developing solution 1 bottle (25 mL / bottle); stop solution 1 bottle (15 mL / bottle); plate sealing film 2 pieces; instruction 1. The kit is stored at 4℃, and the storage period is 6 months. The kit can detect 180 serum samples.

[0060] Method for using the kit:

[0061] 1. Take the kit out of the 4℃ refrigerator, and equilibrate to room temperature. Dilute the 25-fold washing solution to 1-fold using concentration with double distilled water.

[0062] 2. Take out the enzyme-labeled plate, determine the required enzyme-labeled plate strip according to the number of serum samples to be detected, and store the remaining plate strip in a packaging bag at 4℃. The disassembled enzyme-labeled plate is preferably used within 10 days.

[0063] 3. Sample addition: standard positive serum and standard negative serum are each 2 wells, 50 μL / well; the sample to be detected is added to 1 well, 50 μL / well.

[0064] 4. Add monoclonal antibody working solution, 50 μL / well.

[0065] 5. Incubation: seal the enzyme-labeled plate with plate sealing film, and place it in a 37℃ incubator for 90 min.

[0066] 6. Washing: carefully remove the plate sealing film, discard the liquid, and spin dry. Add full washing solution to each well, stand for 30 seconds, and then discard. Repeat 4 times, and pat dry.

[0067] 7. Add HRP labeled goat anti-mouse secondary antibody working solution, 100 μL / well.

[0068] 8. Incubation: Seal the microplate with sealing film and incubate at 37°C for 30 min.

[0069] 9. Washing: Same as step 6.

[0070] 10. Color development: Add 100 μL of TMB color development solution to each well and develop at 37°C in the dark for 15 min.

[0071] 11. Termination: Add stop solution, 50 μL / well.

[0072] 12. Measurement: Immediately measure the absorbance (OD450 value) of each well using an ELISA reader at a wavelength of 450 nm.

[0073] 13. Result Determination

[0074] ① Calculate the OD450 values ​​of the negative and positive control wells and each test serum well on the same ELISA plate. Calculate the inhibition rate (PI) of the standard positive serum, negative serum, and test serum using the following formula: PI = (1 - average OD450 of samples / average OD450 of standard negative serum) × 100%.

[0075] ② The test is valid if the PI of the standard positive serum is >80% and the OD450 of the standard negative serum is >1.0; otherwise, the test result is invalid.

[0076] ③ For the serum sample to be tested, a PI ≥ 27.15% is considered positive; a PI < 27.15% is considered negative.

[0077] The kit's sensitivity, specificity, repeatability, concordance rate, and shelf life were determined.

[0078] Compared with indirect ELISA kits, this invention improves the detection of putrefactive Clostridium perfringens in serum. α Toxin antibodies exhibit higher sensitivity and specificity.

[0079] Clostridium putrefactive standard positive serum was diluted to five levels (1:1, 1:2, 1:14, 1:38, and 1:16). Samples at each dilution were tested using the kit described in this invention to determine the lowest detectable antibody titer. The results showed that the positive serum could still be detected at a dilution of 1:16, indicating that the kit has high sensitivity.

[0080] Detection of Clostridium perfringens using a kit α Positive serum for toxin antibodies β 1 Positive serum for toxin antibodies β 2 Positive serum for toxin antibodies epsilonToxin antibody positive serum, E. coli positive serum and Salmonella positive serum were all negative, indicating that the Clostridium perfringens toxin antigen used in the kit had no cross reaction with other Clostridium toxin of infected sheep and positive serum of common bacteria (E. coli and Salmonella). α The toxin antigen had no cross reaction with other Clostridium toxin of infected sheep and positive serum of common bacteria (E. coli and Salmonella), and the kit had good specificity.

[0081] 20 samples of Clostridium perfringens toxin positive serum and 20 samples of negative serum were prepared. α Toxin positive serum and 20 samples of negative serum were repeatedly detected three times under the same conditions at different times using the same batch of kit, and the determination results were completely the same; the above serum was detected using three batches of kit, and the determination results were completely the same; these results indicated that the kit had good repeatability.

[0082] 50 samples of positive serum and 50 samples of negative serum identified by indirect ELISA kit were detected using the kit to verify the coincidence rate of the kit with the indirect ELISA kit. The results showed that the coincidence rate of the kit with the indirect ELISA kit was 97% (97 / 100).

[0083] The same 20 samples of positive serum and 20 samples of negative serum were detected every 2 weeks using the kit prepared in the same batch. The results showed that the detection results of the kit stored for 6 months on the same serum were the same, indicating that the storage period of the kit was at least 6 months.

[0084] Example 4 Kit detection of field serum samples

[0085] A total of 200 samples of sheep serum collected from 4 farms were detected using the kit in the application, and the detection results are shown in Table 1.

[0086]

[0087] It can be known from the detection results that the coincidence rate of the two methods was 92% (66+118 / 200) when the 200 field serum samples were detected using the kit in the application and the indirect ELISA kit, respectively, indicating that the kit involved in the application can completely replace the indirect ELISA kit to detect Clostridium perfringens alpha toxin serum antibody, and can be popularized and applied in production.

[0088] The above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed in the application, within the spirit and principles of the application, should be covered in the protection scope of the application.

Claims

1. A hybridoma cell line secreting a monoclonal antibody specifically binding to Clostridium perfringens toxin recombinant protein, characterized in that: α Clostridium hybrideum cell line α Toxin-8H2 was deposited at China Center for Type Culture Collection on January 2, 2025, and the deposit number is CCTCC NO: C202535. ​ 2. A monoclonal antibody that specifically binds to Clostridium perfringens α toxin recombinant protein, characterized in that: The heavy chain variable region amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown as SEQ ID NO.

2.

3. The monoclonal antibody of claim 2, which specifically binds to Clostridium perfringens α toxin recombinant protein, characterized by: The monoclonal antibody is produced by the hybridoma cell strain with the preservation number of CCTCC NO: C202535.

4. Use of the monoclonal antibody of claim 2 in the preparation of a reagent or kit for detecting serum antibodies of Clostridium perfringens.

5. A competitive ELISA kit for detecting serum antibodies to Clostridium perfringens in serum, characterized in that: The kit comprises the monoclonal antibody of claim 2.

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