产气荚膜梭菌肠毒素基因原核表达载体的构建

Construction of Prokaryotic Expression Vector of the Enterotoxin Gene of Clostridium perfringens Type C

以含C型产气荚膜梭菌分离株(CP2)肠毒素基因的克隆载体pMD18-T-cpe为材料,根据产气荚膜梭菌开放阅读框设计合成一对特异性引物,采用PCR技术对其进行扩增,经BamHI和EcoRI双酶切后,从胶上回收目的基因,与经过相同两种内切酶处理的原核表达载体pET32a连接,转化大肠杆菌DH5a感受态细胞后,提取质粒进行PCR和BamHI/EcoRI双酶切鉴定后测序。结果表明,肠毒素基因已成功地克隆到原核表达载体上(重组质粒命名为pET32a-cpe),从而构建了产气荚膜梭菌肠毒素基因的原核表达质粒。

A pair of specific primers was designed and synthesized based on the published C. perfringens enterotoxin gene, and amplified the gene from vector pMD18-T-cpe. The PCR products retrieved from 1.0% agarose gel was digested by BamHI and EcoRI and then directly ligased to prokaryotic expression vector pET32a digested by same enzymes. The recombinant plasmid was transferred into competent ceils of E. coli DH5a. The recombinant plasmid was identified by PCR and restriction enzyme and then sequenced. The results showed that the C. perfringens enterotoscin gene was cloned into prokaryotic expression vector pET32a and the recombinant plasmid was named as pET32a-cpe, therefore the prokaryotie expression plasmid of C. perfringens enterotoxin gene was constructed.