结核分枝杆菌Zmp1基因原核表达载体的构建及表达鉴定  

Construction and identification of a prokaryotic expression vector for Zmp1 gene from Mycobacterium tuberculosis

作　　者：张继明[1] 杨春[1] 何永林[1] 穆柳青[1] 张春燕[1] 樊宇[1] 徐蕾[1] 

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

出　　处：《细胞与分子免疫学杂志》2014年第6期573-575,580,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市自然科学基金(cstc2012jjA10023);国家自然科学基金(30771922,30901280)

摘　　要：目的构建结核分枝杆菌锌离子依赖的金属蛋白酶1(Zmp1)基因的原核表达载体,并在大肠杆菌中进行表达。方法以卡介苗(BCG)基因组DNA为模板,采用PCR法扩增Zmp1基因;定向克隆到原核表达载体pET-32a(+)的多克隆位点中,构建重组原核表达质粒pET-32a(+)-Zmp1;转化入大肠杆菌BL21(DE3)中经IPTG诱导表达,表达产物经SDS-PAGE和Western blot法鉴定。结果PCR法扩增出Zmp1基因;重组表达质粒经双酶切及基因测序鉴定构建正确;表达的重组Zmp1融合蛋白相对分子质量(Mr)约为94 000,大小与预期融合蛋白一致;重组Zmp1融合蛋白可与His标签单克隆抗体特异性结合。结论成功构建了Zmp1基因原核表达载体,并在大肠杆菌BL21(DE3)中获得重组Zmp1融合蛋白表达。Objective To construct a prokaryotic expression plasmid for zinc-dependent metalloprotease-1 （Zmpl） gene from Mycobacterium tuberculosis and express the plasmid in E. coil Methods Zmp1 gene was amplified by PCR using the genome of Bacillus Calmette-Guerin vaccine （ BCG ） as a template and inserted into a multiple cloning site of prokaryotic expression vector pET32a（＋）. The constructed prokaryotic expression vector pET32a（＋）-Zmpl was transformed into E. coli BL21 （DE3）, and the recombinant proteins were expressed via IPTG induction. Finally, the expression of Zmp1 protein was detected by SDS-PAGE and Western blotting. Results Restriction analysis and sequencing proved that the recombinant plasmid pET-32a（＋）-Zmpl was constructed correctly. The relative molecular mass of the expressed recombinant protein was about 94 000 which was the same with that of presumed fusion protein. Recombinant Zmpl protein showed a specific binding to monoclonal antibody with His tag. Conclusion The prokaryotic expression vector for Zmpl gene was successfully constructed and the Zmp1 fusion protein was effectively expressed in E. coli BL21 （DE3）.

关 键 词：结核分枝杆菌 Zmp1 原核表达 

分 类 号：R392.33[医药卫生—免疫学] Q786[医药卫生—基础医学]