产气荚膜梭菌青海分离株α毒素基因克隆与表达

Cloning and expression of alpha-toxin gene of Clostridium perfringens Qinghai strain

用PCR技术,从一株田间分离的产气荚膜梭菌基因组中扩增出了1194bp的α毒素基因,经限制性核酸内切酶EcoRⅠ和NcoⅠ双酶切处理后将其连接在经同样内切酶处理的载体pET-28a(+)中的相应位点上,最后转化至受体菌BL21(DE3)中。经PCR、双酶切和核苷酸序列分析,证明重组质粒pET-28a-CPA含有产气荚膜梭菌α毒素基因,再将其转化至大肠杆菌BL21(DE3),经IPTG诱导,目的基因获得了良好表达。以羊抗α毒素多克隆血清进行Western blot分析,抗血清可与该融合蛋白发生特异性反应,表明目的蛋白具有较好的免疫原性。

Alpha-toxin gene was amplified from a field strain of Clostridium peoCringens by polymerase chain reaction （PCR） , PCR product was cleaved with restriction endonucleases EcoR Ⅰ and Nco Ⅰ , and inserted into the EcoR Ⅰ/Nco Ⅰ site of pET-28a（ ＋ ）vector. Recombinant pET-28a-CPA plasmid was transformed into BL21 （DE3）cells. The obtained plasmids were studied in detail by PCR, restriction endonuclease analysis and nucleotide sequencing. The recombinant plasmid was transformed into E. coli BI21 （ DE3 ） and expressed under the induction of IPTG. Western blot analysis indicated that polyclonal goat-anti-serum against alpha-toxin had specific reaction with recombinant protein. These results indicated that the target protein had a good immunogenicity.