结核分枝杆菌RD1区PPE68/GST融合蛋白表达质粒的构建及表达

Construction and expression of Mycobacterium tuberculosis PPE68/GST fusion protein expression plasmid

目的:构建结核分枝杆菌PPE68/GST融合蛋白表达质粒,并在大肠杆菌JM109中诱导表达.方法:利用PCR技术从结核杆菌H37Rv中扩增Rv3873基因,并将其定向克隆pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-Rv3873并转化E.coliJM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.采用GST蛋白纯化试剂盒进行重组融合蛋白纯化,SDS-PAGE和WesternBlot鉴定重组表达产物.结果:成功构建原核表达质粒pGEX-4T-1-Rv3873并表达出约Mr63000大小的PPE68/GST融合蛋白,占总菌体的40%.WesternBlot检测该融合蛋白能和结核患者多价抗血清发生反应.结论:成功地构建了原核表达质粒pGEX-4T-1-Rv3873,并在大肠杆菌得到表达.

AIM： To construct the Mycobacterium tubereulosis （MTB） PPE68/GST fusion protein expression vector and induce its expression in E. coil JM109. METHODS： Rv3873 gene was amplified from MTB H37Rv by PCR and directionally cloned into expression plasmid pGEX-gT-1. Construct the recombinant expression plasmid pGEX-4T-1-Rv3873 and express it in E. coli JM109 under the induction of isopropyl-13-D-thiogalactopyranoside （IPTG）. The fusion protein was purified by GST affinity chromatgraphy and confirmed by SDS-PAGE and Western Blot. RESULTS： The desired PPE68/GST fusion protein （M63 000） was successfully expressed, accounting for 40% of the total bacteria. Western bolt revealed that the fusion protein could react with the polyvalent antiserum of tuberculosis patients. CONCLUSION： Prokaryotic expression plasmid pGEX-4T-1-Rv3873 was successfully cloned and expressed in E. coil JM109.