结核分枝杆菌ESAT6基因克隆与表达质粒构建

Construction of the recombinant expression plasmid encoding ESAT6 gene of the tuberculosis bacterium

目的 克隆结核分枝杆菌 (MTB)分泌蛋白ESAT6基因 ,并构建重组表达质粒。方法 根据Genbank中ESAT6基因序列 ,针对其编码区合成引物 ,采用PCR方法从结核分枝杆菌H37Rv基因组DNA中扩增出ESAT6基因 ,并连接到T载体 ,然后定向克隆到原核表达质粒pGEX 4T 2 ,阳性克隆用酶切和DNA测序鉴定。结果 经双内切酶消化所切下的片段 ,大小与预计相符 ;测序结果证实基因序列正确 ,符合表达框架。结论 成功构建了重组原核表达质粒 pGEX ESAT6。

Objective To clone and construct a expression plasmid containing ESAT6 gene of tuberculosis bacterium.Methods According to the nucleic acid sequence of the ESAT6 gene from Genbank,a pair of primer was synthesized and ESAT6 gene was amplified (PCR method) from the H37Rv,then cloned into pGEM T easy vector,finally,subcloned into pGEX 4T 2 according to its special orientation.The positive clone was identified by restriction endoenzyme analysis and DNA sequencing.Results The fragment digested by endoenzymes was as large as the predicted result.The sequence was the same as the sequence reported on the literatures.Conclusion The ESAT6 gene was successfully cloned into pGEX 4T 2 pronucleus expression vector.