结核分枝杆菌ATP依赖的丝氨酸蛋白酶调节亚基C2基因原核表达质粒的构建及表达  

Construction and expression of prokaryotic expression vector for ATP dependent Clp regulatory subunit C2 gene of Mycobacterium tuberculosis

作　　者：穆柳青[1,2] 李岱容[3] 张春燕[1,2] 杨静[1,2] 冯鑫[1,2] 杨春[1,2] 

机构地区：[1]重庆医科大学病原生物学教研室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆400016 [3]重庆医科大学附属第一医院呼吸科,重庆400016

出　　处：《中国生物制品学杂志》2013年第10期1392-1394,1399,共4页Chinese Journal of Biologicals

基　　金：国家青年科学基金(81101216)

摘　　要：目的构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)ATP依赖的丝氨酸蛋白酶调节亚基C2(ATP-dependent Clp regulatory subunit C2,ClpC2)基因的原核表达质粒,并在大肠埃希菌中表达重组蛋白。方法以MTB H37Rv基因组DNA为模板,PCR扩增ClpC2基因,插入表达载体pET32a(+)中,构建重组原核表达质粒pET32a(+)-ClpC2,经双酶切及测序鉴定正确后,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切和测序鉴定,证明构建正确;表达的重组融合蛋白相对分子质量约46 000,可与鼠抗组氨酸单克隆抗体特异性结合。结论已成功构建了重组表达质粒pET32a(+)-ClpC2,并在大肠埃希菌中表达了重组蛋白,为进一步研究ClpC2蛋白的生物学功能奠定了基础。Objective To construct the prokaryotic expression vector for ATP-dependent Clp regulatory subunit C2 （ClpC2） gene of Mycobacterium tuberculosis （MTB） and express in E. coll. Methods CIpC2 gene was amplified by PCR using the genomic DNA of MTB H37Rv strain as a template, and inserted into expression vector pET32a （＋）. The constructed recombinant plasmid pET32a （＋）-ClpC2 was identified by restriction analysis and sequencing, then trans- formed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pET32a （＋）-CIpC2 was constructed correctly. The expressed fusion protein, with a relative molecular mass of about 46 000, showed specific binding to mouse monoclonal antibody against histidine. Conclusion Recombinant plasmid pET32a （＋）-CIpC2 was suc- cessfully constructed and expressed in E. coli, which laid a tbundation of further study on biological function of ClpC2 protein.

关 键 词：结核分枝杆菌 ClpC2基因 原核细胞 基因表达 

分 类 号：R378.911[医药卫生—病原生物学] Q782[医药卫生—基础医学]