Solation And Identification Of Yanbian Strain Clostridium Chauvoei, And Prokaryotic Expression Of Its CCTA Gene

Emphysematous gangrene caused by Clostridium chauvoei is an acute,thermal resistance, septic infectious diseases. The characteristic of its fast onset, high mortality has brought severe damage to animal husbandry. The disease typically manifests in clinic with occurrence of gas, inflammatory swelling in subcutaneous tissue and muscle fullness site, which push lesion with crepitus. After 2006, outbreak of bovine Emphysematous gangrene was reported successively in Dunhua City, Yilan and Sandao town in Yanbian, which constrain economic development of the local animal husbandry. With population infection increasing, Emphysematous gangrene probably threat to human security.In order to preparation safe and efficient novel vaccine of emphysema gangrene to control the occurrence of Emphysematous gangrene disease in Yanbian, we collect-ed liver, spleen, muscle tissue from suspected Yanbian cattle, and isolated and identif-ied Yanbian strain. According to Clostridium chauvoei cctA gene published in GenBa-nk, a pair of specific primers were designed and synthesized. cctA gene was amplified by PCR using total DNA of Yanbian strain Clostridium chauvoei. After cloning and sequencing, we analysed physical and chemical properties of cytotoxic CctA protein, and predicted its structure and function domain, protein secondary structure and ter-tiary structure with antigen epitope parameters and so on using bioinformatics met-hods. The cloned plasmid pMD 19-T-cctA was digested with BamH I and Xho I and connected with the prokaryotic expression vector pEGX-4T-1, then constructed prokaryotic expression plasmid of pGEX-4T-cctA. We screened positive clones and made an inducible expression. The expressed protein was identified by SDS-PAGE and Western-blot.The result showed that Yanbian strain C. chauvoei were separated successfully. Through comparing to the reference sequence (JQ692583), three mutations was identified, the sequence homologies of nucleotides and amino acids were both 99.0%. Theoretical isoelectric point of protein cctA without the transmembrane region and signal peptide sequence was 6.79. Secondary structure of protein cctA mainly is beta-folding and random curl and were predicted 5 main antigenic epitope regions. The 44 kDa domain of expressed protein was identified by SDS-PAGE and Western blot, which indicated that the domain has a better reactionogenicity.In conclusion, our study showed the specificity protein was identified might provide a useful and reliable reference basic to Yanbian strain C.chauvoei diagnosis and further analysis cctA protein function. The results also will pave the way for future the preparation of mono-cloned antibody and the development synthetic peptide vaccine of Clostridium chauvoei.