结核分枝杆菌重组质粒pGEX-ESAT-6构建及其在大肠埃希菌BL21(DE3)中表达效率的研究

Construction and expression efficiency of recombinant plasmid pGEX-ESAT-6 of Mycobacterium tuberculosis

目的构建结核分枝杆菌(MTB)重组质粒pGEX-ESAT-6,分析ESAT-6抗原在大肠埃希菌BL21(DE3)中的表达效率。方法以结核分枝杆菌H37Rv标准株基因组DNA为模板,通过PCR扩增得到ESAT-6抗原编码基因;将该基因定向克隆于含有谷胱甘肽-S-转移酶(GST)基因的高效原核表达载体pGEX-1λT,经酶切鉴定后以IPTG诱导表达ESAT-6/GST融合蛋白;SDS-PAGE及Western blot对表达产物进行鉴定。结果PCR扩增出288bp的ESAT-6基因;双酶切证实ESAT6基因成功插入pGEX-1λT中,构建了pGEX-ESAT-6穿梭表达载体。SDS-PAGE分析重组质粒pGEX-ESAT-6表达产物的分子质量单位为35ku,表达效率为20%,Western blot检测该蛋白能被活动性结核病人血清特异识别。结论结核分枝杆菌重组质粒pGEX-ESAT-6在大肠埃希菌中获得了高效融合表达,表达的ESAT-6重组蛋白具有抗原特异性。

Objective To investigate expression efficiency of the recombinant plasmid pGEX ESAT-6 of Mycobacterium tuberculosis in Escherichia coli BL21（DE3）. Methods ESAT-6 antigen gene were amplified by PCR. Then the gene was cloned into prokaryotic expression vector pGEX-1λT containing gluathione-S-transferaze（GST） gene. The recombi- nant plasmid （pGEX-ESAT-6） was analyzed with restriction-endonuclease digestion. The expression of pGEX-ESAT-6 was induced with isopropyl-β-D-thiogalactopyranosid （IPTG） and protein ESAT-6 /GST was examined by SDS-PAGE and Western blot techniques. Results 288 bp gene of ESAT-6 was amplified by PCR and cloned into pGEX-1λT by restriction analysis, the recombinant plasmid pGEX-ESAT-6 was constructed. It was demonstrated with SDS-PAGE and Western blot that the protein ESAT-6 /GST were expressed in E. coli BL21 （DE3）. Conclusion The gene ESAT-6 of M. tuberculosis was highly expressed in E. coli in fused form with GST and this kind of protein shows specific antigenicity.