结核分枝杆菌Mtb8.4-Hspx和Hspx-Mtb8.4重组基因克隆及表达  

Cloning and expression of recombinant gene MH(Mtb8.4-Hspx) and HM(Hspx-Mtb8.4) without affinity tags from Mycobacterial tuberculosis

作　　者：吴玉敏[1,2] 谭继英[1,2] 王革[3] 刘万波[1,2] 胡丽娜[3] 祝秉东[1,4] 

机构地区：[1]兰州大学结核病研究中心,甘肃兰州730000 [2]兰州大学基础医学院免疫学研究所 [3]兰州生物制品研究所 [4]兰州大学基础医学院病原生物学研究所

出　　处：《中国病原生物学杂志》2010年第6期414-417,共4页Journal of Pathogen Biology

基　　金：卫生部艾滋病和病毒性肝炎等重大传染病防治科技重大专项(No.2008zx100030135);兰州大学医学科研基金资助项目(LZUYX200704)

摘　　要：目的构建结核分枝杆菌去标签重组蛋白Mtb8.4-Hspx(MH)和Hspx-Mtb8.4(HM)原核表达载体,并在大肠埃希菌株BL21(DE3)中表达。方法以结核分枝杆菌H37Rv基因组DNA为模板,PCR扩增Mtb8.4、Hspx基因,产物经纯化后与载体PET30a连接、转化及测序鉴定,构建原核重组表达质粒PET30a-Mtb8.4和PET30a-Hspx。抽提重组质粒,产物经纯化后与目的基因Hspx、Mtb8.4连接,转化及测序鉴定,构建重组目的基因质粒PET30a-Mtb8.4-Hspx(PET30a-MH)和PET30a-Hspx-Mtb8.4(PET30a-HM)载体,转化大肠埃希菌BL21,以IPTG诱导,SDS-PAGE电泳检测表达产物。结果 SDS-PAGE蛋白电泳验证去标签MH和HM融合蛋白分子质量单位为28 ku,与预期值相符。结论本研究成功表达去标签结核杆菌重组蛋白MH和HM,为结核亚单位疫苗免疫原性和临床前研究奠定了基础。Objective To construct recombinant protein Mtb8.4-Hspx（MH） and Hspx-Mtb8.4（HM） without affinity tags and express them in the Escherichia coli BL21（DE3） strain.Methods The two genes encoding proteins Mtb8.4 and Hspx were amplified based on the genome DNA sequence of H37Rv by PCR and were then purified and cloned into prokaryotic expression vector PET30a to construct recombinant plasmids PET30a-Mtb8.4 and PET30a-Hspx.The recombinant plasmids（PET30a-Mtb8.4 and PET30a-Hspx） were extracted and purified and then Hspx and Mtb8.4 were respectively inserted to construct recombinant plasmids PET30a-Mtb8.4-Hspx（PET30a-MH） and PET30a-Hspx-Mtb8.4（PET30a-HM）.The recombinant plasmids were retransformed into the E.coli BL21 strain and were then induced by IPTG and identified by SDS-PAGE.Results The recombinant proteins MH and HM without affinity tags had an apparent molecular weight of 28 ku,which is consistent with the predicted value.Conclusion The successful expression of MH and HM proteins without affinity tags has provided grounds for the immunogenicity of a Mycobacterium Tuberculosis subunit vaccine and the preclinical study of that vaccine.

关 键 词：分枝杆菌 结核 MTB8.4 HSPX 克隆 表达 

分 类 号：R378.911[医药卫生—病原生物学]