Preparation Of The Vaccine Against Cervus Hemorrhagicenteritis Of Shanxi Province And Nucleotide Sequencing Of The Beta-toxin Of Clostridium Perfringens

In this study, 26 Clostridium perfringens type A and 6 Clostridium perfringens type C were identified from viscus and intestinal contents of the dead cervus of 32 cases caught hemorrhagic enteritis in Shanxi Province. T5-A and J28-C which were strongest virulence were selected from isolates of Clostridium perfringens type A and C. The best conditions of producing toxin of Clostridium perfringens were cultured 7 hours in 35D in anaerobic beef pepsin digest broth and anaerobic environment. T5-A and J28-C strains were cultured in this conditions, then they were inactivated with 0.5%,0.7%,0.9% formaldehyde. Five days later, the cultures were inactivated. Two types of cultures were mixed with 1:1. Then 10% Al(OH)3 was added into the qualified liquid and stood for 10d at 2 to 8â–¡, then moved away pure liquid of 50 percent of the gross volume, shook and divided into bottles. The dose of formaldehyde is 0.5% by the results of safety test, and the least dose of immunity is 2ml which was determinated by Challenged test. And the valences of antibody were determinated at different dose of immunity. The immune duration and the valences of antibodies of multivalent inactive vaccines were determinated at 3,6,9 months. The result suggest that the decrease of the valences of antibody of Clostridium perfringens type C is slow,it can protect 75% even at 9 months; while Clostridium perfringens type A is object, it can protect 75% at 6 months. The major toxin of Clostridium perfringens type C was beta-toxin. In this study, the recombinant plasmid of beta-toxin gene was made then beta-toxin gene was cloned and nucleotide sequence was determined. The results were agreement with the standard gene sequence ofbeta-toxin and lay a good foundation for the preparation of gene vaccine.