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

作　　者：曾林子[1] 陈建平[2] 刘成君[1] 李红霞[2] 姚卫[3] 杨志荣[1] 

机构地区：[1]四川大学生物资源与生态环境教育部重点实验室 [2]四川大学华西医学中心基础医学与法医学院寄生虫教研室,四川成都610052 [3]四川大学绵阳市疾病预防控制中心,四川绵阳621000

出　　处：《第四军医大学学报》2007年第1期59-61,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30300302);四川省学术和技术带头人培养基金(4200316)

摘　　要：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.

关 键 词：结核分枝杆菌 RD1区 PPE68/GST融合蛋白 表达 

分 类 号：R392.11[医药卫生—免疫学]