CFP10-ESAT6-PPE68融合基因原核表达质粒的构建和表达  

Construction and expression of prokaryotic expression vector of CFP10-ESAT6-PPE68 fusion gene of mycobacterium tuberculosis

作　　者：冯鑫 杨静[1] 张春燕 穆柳青 徐蕾 何永林[1] 董志玲 杨春[1] 

机构地区：[1]重庆医科大学病原生物学教研室、重庆医科大学分子医学与肿瘤研究中心,重庆400016

出　　处：《重庆医科大学学报》2013年第4期408-412,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30901280);重庆市自然科学基金资助项目(编号:cstc2012jjA10023)

摘　　要：目的:构建结核分枝杆菌(mycobacterium tuberculosis,M.tb)CFP10-ESAT6-PPE68融合基因原核表达质粒,并且在大肠杆菌中表达融合蛋白。方法:以M.tb H37Rv株DNA作为模板,PCR法扩增ESAT6和PPE68基因,利用基因拼接技术中的Gene-SOEing法扩增ESAT6-PPE68融合基因,克隆至原核表达质粒pET-32a(+)中,构建重组表达质粒pET-32a(+)-ESAT6-PPE68。再分别以H37Rv株DNA和重组质粒pET-32a(+)-ESAT6-PPE68为模板扩增CFP10和ESAT6-PPE68基因,Gene-SOEing法获取目的基因CFP10-ESAT6-PPE68,克隆至pET-32a(+)载体,构建重组表达质粒pET-32a(+)-CFP10-ESAT6-PPE68,转化入大肠杆菌BL21(DE3),IPTG诱导表达。结果:CFP10-ESAT6-PPE68酶切片段大小与理论值符合,基因测序结果显示融合基因CFP10-ESAT6-PPE68的序列与Genbank中序列一致,并在大肠杆菌中成功诱导表达了目的蛋白,经SDS-PAGE分析和Western blot鉴定,相对分子量约为77 kD。结论:成功构建了M.tb CFP10-ESAT6-PPE68融合基因的原核表达质粒,并诱导表达出了融合蛋白,为该融合蛋白在结核病血清诊断学中的应用奠定了实验基础。Objective：To construct a prokaryotic expression vector of culture filtrate protein 10（CFP10）-early secreted antigenic target6 kD Antigen（ESAT6）-pentose-5-phosphate-3-epimerase 68（PPE68） fusion gene of mycobacterium tuberculosis（M.tb） and to express in escherichia coli（E.coli）.Methods：ESAT6 and PPE68 genes were amplified by PCR respectively using the genomic DNA of M.tb H37Rv strain as template.Fusion gene ESAT6-PPE68 was obtained by Gene-SOEing（gene splicing by over lap extension）,and then was cloned into pET-32a（＋）.CFP10 and ESAT6-PPE68 genes were amplified from genomic DNA of M.tb H37Rv strain and recombinant plasmid pET-32a（＋）-ESAT6-PPE68 was constructed by Gene-SOEing as well.Constructed recombinant plasmid pET-32a（＋）-CFP10-ESAT6-PPE68 was transformed into E.coli BL21（DE3） and its expressions were induced with IPTG.Results： Restriction enzyme analysis and sequencing proved that recombinant plasmid pET-32a（＋）-CFP10-ESAT6-PPE68 was constructed correctly.Expressed fusion protein with a relative molecular mass of 77 000 was identified by SDS-PAGE and Western blot.Conclusions：Prokaryotic expression vector of CFP10-ESAT6-PPE68 fusion gene of M.tb is constructed successfully and the fusion protein is expressed in E.coli BL21（DE3）,which lay a foundation for applying the fusion protein in serological diagnosis of tuberculosis in the further.

关 键 词：结核分枝杆菌 培养滤液蛋白10 6kD早期分泌抗原靶 戊糖-5-磷酸-3差向异构酶 融合蛋白 

分 类 号：R37[医药卫生—病原生物学]