Preparation Of C,D Clostridium Perfringens Toxoid And Recombinant Clostridium Septicum Alpha Toxin Bivalent Vaccine And Evaluation Of Its Immune Effect

Clostridiasis is a general term for diseases caused by pathogenic bacteria in the Clostridium genus.These usually include Clostridium perfringens,Clostridium septicum,Clostridium tetanus,etc.Among them,the most threatening to China is Clostridium perfringens and Clostridium septicum.Clostridiasis has a rapid onset,a short course,and a high mortality rate.Animals often die as soon as they become ill.Therefore,immunization is the most direct and effective way to prevent this disease.At present,the main antigenic components of triple quadruple vaccines in China are bacterial cells or toxoids.As a strict anaerobic bacterium,Clostridium septicum is difficult to cultivate and its toxicity is weak,which greatly reduces the protective ability of the vaccine.Recombinant Clostridium Septicum Alpha Toxin expressed through genetic engineering can have the immunogenicity of natural toxins,and the production process is simple,which can be used to upgrade the triple-quad prevention vaccine.Therefore,this study intends to prepare bivalent vaccines of Clostridium perfringens C and D and Clostridium septicum.In this study,Clostridium perfringens type C(NCTC3180)and Clostridium perfringens type D(NCTC 8346)were resuscitated,enriched,and toxic.Filter the bacteria with a disposable bacteria filter to obtain a toxin solution.A recombinant protein expressing the recombinant alpha toxin of Clostridium septicum was constructed using our laboratory to produce a recombinant protein through a prokaryotic expression system.The C and D type Clostridium perfringens natural toxin and Clostridium putrefaciens recombinant alpha toxin are mixed in a certain ratio,and after formaldehyde inactivation,a white oil adjuvant is added to prepare a bivalent bivalent vaccine.Physical properties,sterility,safety and animal protection tests were performed on the prepared vaccine.The results show that the toxin prepared above can be completely inactivated after being inactivated with formaldehyde at a final concentration of 0.3% for 96 hours,and can maintain good antigenicity.The test results of the prepared bivalent bivalent vaccine meet the quality standards of veterinary biological products of the People's Republic of China.After immunizing mice with 0.2mL vaccine,the protection rate can reach 100%.21 days after inoculation of 5 mL vaccine to 6-7 month old sheep,the antibody titer level in sheep serum was evaluated by indirect ELISA and toxin neutralization test.It is 1: 3200,the antibody titer against Clostridium perfringens toxin type D is 1: 6400,and the antibody titer against Clostridium perfringens is 1: 6400.The neutralization titers are 400,800 and 800 respectively.According to the provisions of the third part of the 2010 edition of the Chinese veterinary medicine code,if the neutralization titer of the serum reaches 1 for the fast epidemic(i.e.0.1 ml immune animal serum can neutralize 1 MLD toxin)and 3 for the enterotoxemia(i.e.0.1 ml immune animal serum can neutralize 3 MLD toxin),the vaccine is qualified.This shows that the vaccine prepared in this experiment has high titer,is safe and reliable,and can be used for the prevention of clostridial disease against ruminants such as cattle and sheep.