| Clostridium perfringens C.perfringens, known as Clostridium welchii is widely distributed in nature and the animal intestinal bacteria, belonging to conditional pathogenic bacteria. It can produce a variety of toxins and enzymes. The main toxin is alpha, beta,epsilon, and iota, and the beta toxin has strong neurotoxicity, cell toxicity and lethality. It can cause Struck calves, sheep, lamb, piglet intestinal toxemia, and human and animal necrotic enteritis. Rapid morbidity and mortality in livestock infection.Therefore, it is called "sudden death of livestock". A variety of livestock and wild animals, especially within 3weeks of young animals are more susceptible to infection, causing serious losses to the livestock industry in china. In the mid 1980 s, China was once widespread and serious outbreak, due to antibiotic use, the epidemic of the disease to a certain degree of inhibition.Sometimes clinical symptoms was not obvious, but there were still a large number of reports. Ministry of agriculture will be listed as two categories of infectious diseases.The main purpose of this study is relatively pure Clostridium perfringens toxin preparation using monoclonal antibody. B, C-type Clostridium perfringens toxin produced major lethal toxins, but C-type bacteria also produced small amount of alpha toxin.This study was the first to adopt Sephadex G-25 crude extract of Clostridium perfringens toxins desalting, and then through the Sephadex G-200 of concentrated liquid of purification methods, finally got a relatively pure beta toxin. We purified the beta toxin by polyacrylamide gel electrophoresis(SDS-PAGE) to detect the purity of the isolated protein.Therefore, the purified protein has a very important role in the preparation of monoclonal antibodies.In this study, the natural purified beta toxin was used to carry out immunization of BALB/c mice of 6-8 weeks old, and the spleen cells of immunized mice were immunized 3days to get the fusion of Sp2/0 cells and myeloma cells. After screened by indirect ELISA and limited dilution subcloning method obtained the 2 strains of stable secretion in type C perfringens beta toxin of Clostridium perfringens positive hybridoma cell lines. The growth state was injected into the abdominal cavity of mice, and the ascites were collected after abdominal enlargement.Lastely, we used the method of caprylic acid ammonium sulfate purification to obtain monoclonal antibodies against Clostridium perfringens toxin.In this study, we successfully obtained two hybrid tumor cells,named as G7, F4.Indirect ELISA method known to secrete antibodies against the two strains of anti- beta toxin showed that the antibody titer of monoclonal antibody titer in mouse ascites was significantly higher than that of the culture supernatant of hybridoma cells, and the maximum can reach 1:102400Western Blot analysis showed that the two monoclonal antibodies were able to react with the purified beta toxin, but not with other toxins.Monoclonal antibody has the advantages of single biological activity, high purity, and strong specificity of binding to antigen. We can use monoclonal antibody to detect diseases and treat diseases quickly. The successful preparation of against beta toxin monoclonal antibody by polyacrylamide gel electrophoresis and immunoblot experiments to further validate the monoclonal antibody of against beta toxin of purity and immunogenicity. It is important to lay the foundation for the rapid construction of effective method for detection of Clostridium perfringens type of toxin in the next step. This method has a good application prospect in the field of rapid detection of disease, food safety and so on. |
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