大肠杆菌表达腐败梭菌α毒素及其产物的生物学特性鉴定

Expression of Alpha Toxin Gene of Clostridium septicum in E. coli and its Biological Characteristics

为了制备大肠杆菌源的重组腐败梭菌α毒素并对其生物学特性鉴定。利用PCR扩增α毒素基因,构建重组表达质粒pET32a-csa,转化E.coli BL21(DE3),IPTG诱导表达,对产物的反应原性、溶血活性、细胞毒性、小鼠致死毒性、毒力和毒性中和等生物学特性鉴定。结果显示,重组α毒素主要表达形式为包涵体,产物能够与腐败梭菌抗毒素血清反应,不能使绵羊红细胞发生溶血,具有细胞毒性和小鼠致死毒性,毒力超过300 MLD/mL,而且这种毒性可以被抗毒素中和。结果表明,通过大肠杆菌原核表达可以制备具有一定生物活性的重组腐败梭菌α毒素。

In order to prepare recombinant alpha toxin from Clostridium septicum and study its biological characteristics,the recombinant expression plasmid vector pET32a-csa was constructed and transformed into E. coli BL21,and the recombinant alpha toxin was expressed, Then,we studied its reactionogenicity, cytotoxicity, toxicity, hemolytic activity and neutralization tests. The results showed that the recombinant alpha toxin was effectively expressed mainly in the inclusion bodies. And the result of Western-blot test showed that the recombinant alpha toxin specially reacted with the antitoxin of Clostridium septicum. The recombinant alpha toxin caused hemolysis. The mouse tests confirmed that each milliliter of the recombinant alpha toxin contained more than 300 MLD, and its toxicity was neutralized by the antitoxin of Clostridium septicum. The results indicated that the E. coli prokaryotic expression can be used to prepare the activated recombinant alpha toxin of Clostridium septicum.