Isolation And Identification Of Clostridium Perfringens From Cattle And Prokaryotic Expression Of ?,?₂,? Toxin Genes

Clostridium perfringens in cattle is mainly caused by Clostridium perfringens type A,C,and D,it is extremely harmful to the animal husbandry,but there is no effective control measure.In this study,the ?-toxin gene of Clostridium perfringens type A,the ?2-toxin gene of Clostridium perfringens type C,and the ?-toxin gene of Clostridium perfringens type D were expressed by prokaryotes,it laid the foundation for making genetically engineered vaccines.Clinically isolated and identified 17 strains of Clostridium perfringens of bovine origin.After single factor toxin ELISA and multiple PCR typing,10 strains of Clostridium perfringens type A and 4 strains of clostridium perfringens type C were finally identified,3 strains of Clostridium perfringens type D.The strain with strong virulence was selected by half lethal dose test in mice for follow-up experiment.The ?,?₂ and ? toxin gene fragments were cloned to construct cloning vectors pMD18-T-?,pMD18-T-?₂ and pMD18-T-?.Insert the identified gene fragments into the prokaryotic expression vector pET-32a?+?to construct the prokaryotic expression vectors pET-32a?+?-?,pET-32a?+?-?₂ and pET-32a?+?-s,introduce the constructed recombinant expression vector into E.coli BL21?DE3?strain,induced by IPTG,and detect the reactogenicity of the expressed protein by Western blot.The results showed that the cloning vectors pMD18-T-?,pMD18-T-?₂ and pMD18-T-?were successfully constructed,and the target gene fragments were 1092 bp,987 bp and 798 bp,respectively.Recombinant strains pET-32a?+?-?/BL21?DE3?,pET-32a?+?-?₂/BL21?DE3?and pET-32a?+?-?/BL21?DE3?at 37?,1mmol/L IPTG induced good expression.SDS-PAGE analysis showed that the expressed proteins were 41 kDa,27 kDa,and 32 kDa,respectively,which were consistent with the expected size.The results of Western blot showed that the expressed a toxin protein,?₂ toxin protein and s toxin protein had good reactivity.In summary,this study successfully constructed pET-32a?+?-?/BL21?DE3?,pET-32a?+?-?₂/BL21?DE3?and pET-32a?+?-?/BL21?DE3?The three recombinant strains can express a toxin protein,?₂ toxin protein and ? toxin protein well.Western blot test shows that the three toxin proteins expressed can react with specific antibodies and have good reactogenicity.