重组结核分枝杆菌ESAT6-PPE68融合蛋白的制备  

Preparation of recombinant Mycobacterium tuberculosis ESAT6-PPE68 fusion protein

作　　者：李红霞[1] 陈建平[1] 曾林子[2] 刘刚[3] 姚卫[4] 杨筠[3] 刘杨椅[3] 王涛 田玉[1] 

机构地区：[1]四川大学华西医学中心基础医学与法医学院寄生虫学教研室,病原生物学实验室,四川成都610041 [2]四川大学生命科学学院,四川成都610041 [3]四川省疾病预防控制中心结核病预防控制所,四川成都610041 [4]四川绵阳市疾病预防控制中心,四川绵阳621000

出　　处：《南方医科大学学报》2007年第2期131-135,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(39870656);四川省学术和技术带头人培养基金(4200316)~~

摘　　要：目的构建结核分支杆菌esat6-ppe68融合基因及其原核表达载体,在大肠杆菌中表达融合蛋白ESAT6-PPE68。方法用基因拼接(GeneSOEing)法扩增esat6-ppe68融合基因,并将其定向克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pGesat6-ppe68。重组子经限制性内切酶分析、聚合酶链式反应及测序鉴定后转化宿主菌大肠杆菌BL21,IPTG(异丙基-β-D硫代半乳糖苷)诱导表达约69000带谷胱苷肽硫转移酶蛋白(GST)标签的rESAT6-PPE68融合蛋白,经谷胱苷肽-硫-转移酶融合蛋白纯化试剂盒得到纯化的融合蛋白,产物进行SDS-PAGE电泳、Western-blotting鉴定。结果重组质粒pGesat6-ppe68中目的基因测序结果与报道序列完全相同;在大肠杆菌中以可溶性非包涵体形式表达;表达量约占菌体总蛋白的40%,表达蛋白纯化后获得纯度为90%左右的重组蛋白;Westernblotting结果证实重组蛋白与确诊的结核病患者血清发生特异免疫反应。结论成功构建了原核表达载体pGesat6-ppe68,获得了rESAT6-PPE68融合蛋白,为rESAT6-PPE68融合蛋白在结核病诊断中的应用奠定了基础。Objective To construct esat6-ppe68 fusion gene and its prokaryotic expression vector for expression in E.coli. Methods With GeneSOEing method, a fusion gene was constructed by splicing esat6 gene and ppe68 gene and cloned into pGEX-4T-1 plasmid to construct the recombinant prokaryotic expression plasmid pGesat6-ppe68. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis of the plasmid, E. coli BL21 was transformed with the recombinant plasmid and induced with IPTG to obtain the expression of the fusion protein ESAT6-PPE68 with GST-tag （about 69 kD）, which were purified with GST-fusion protein purification kit. The expression ofesat6-ppe68 fusion gene was subsequently detected by SDS-PAGE and Western blot analysis. Results The sequence ofesat6 and ppe68 in the recombinant plasmid was consistent with that in GenBank report. The fusion protein was detected in the cytoplasm in soluble form and represented approximately 40% of the total bacterial protein of E.coli. After purification, the purity of the fusion protein reached 90%, and its antigenicity was confirmed by Western blotting. Conclusion The prokaryotic expression vector pGesat6-ppe68 has been constructed and the fusion protein ESAT6-PPE68 obtained successfully, which provides an experimental basis for potential application of the recombinant ESAT6-PPE68 in the diagnosis of tuberculosis.

关 键 词：结核分枝杆菌 esat6-ppe68融合基因 rESAT6-PPE68融合蛋白 原核表达 

分 类 号：R346[医药卫生—基础医学]