含产气荚膜梭菌肠毒素基因重组质粒pET-28a-CPE的构建和表达

Construction and expression of the recombinant prokaryotic plasmid pET-28a-CPE

目的构建含全长产气荚膜梭菌肠毒素(CPE)基因的重组表达质粒pET-28a-CPE并进行原核表达。方法培养并提取表达肠毒素的产气荚膜梭菌标准菌株64615的基因组DNA,PCR扩增出全长CPE基因片段并连接入pET-28a载体质粒中,构建重组原核表达质粒pET-28a-CPE,进行酶切及测序鉴定,并转化入感受态大肠杆菌(E.coli)BL21中,用IPTG诱导CPE表达。结果复苏、培养成功产肠毒素的产气荚膜梭菌标准菌株64615,成功构建了表达质粒pET-28a-CPE,经酶切及测序鉴定结果与设计的完全相符,成功用E.coli BL21进行了原核表达,目的蛋白CPE占总表达蛋白45.37%。结论本实验成功构建并表达了含CPE的重组质粒pET-28a-CPE,为进一步观察CPE对前列腺肿瘤等的生物学作用奠定了基础。

Objective To construct and express a recombinant prokaryotic plasmid pET 28a CPE. Methods The standard Clos tridium perfringens 64615 strain which could produce Clostridium perfringens enterotoxin （CPE） was incubated and identified. The full length gene of CPE was extracted and amplified from the whole length DNA of the CI. Perfringens,and it was correctly subcloned into the vector pET 28a by restriction endonuclease digestion with NdeI plus XhoI. After inverting into competent E. coli BL21 ,recombinant plasmid pET 28a-CPE was identified by DNA sequencing and expressed induced by IPTG. The expression of CPE was identified by SDS-PAGE. Results The plasmid pET 28a CPE was successfully constructed and confirmed by DNA se quencing. The CPE was expressed in E. coli BL21,and was about 45.37%. Conclusion With DNA recombination techniques,the prokaryotic expression plasmid which includes the CPE gene was cloned and expressed successsfully, which offers a basis for investigating CPE＇s biological effect on tumors of prostate.