结核分枝杆菌毒力基因片段Rv0450c分子克隆及原核表达

Molecuar cloning of Rv0405c virulence gene in Mycobacterium tuberculosis and its expression in Escherichia coli

目的通过分子克隆的方法对结核分枝杆菌H37Rv中编码MMPL4蛋白的Rv0450c基因以及编码该蛋白一个161氨基酸的膜内区域(I161)和一个371氨基酸的膜外区域(O371)的基因片段在大肠杆菌中的表达和纯化方法进行研究,为进一步探讨MMPL4与结核分枝杆菌耐药相关功能性研究以及抗体制备提供依据。方法以结核分枝杆菌H37Rv基因组DNA为模板,采用PCR方法扩增Rv0450c基因片段,并克隆到原核表达载体pET28b质粒中,获得的重组质粒转化原核表达宿主大肠杆菌BL2 1(DE3)pLysS中,经IPTG诱导后SDS-PAGE电泳方法检测重组蛋白的表达,并进行纯化。结果从结核分枝杆菌基因组H37RvDNA中成功扩增出Rv0450c基因及编码I161/O371的DNA片段,并构建原核表达质粒pET28b-Rv0450c,pET28b-I161和pET28b-O371重组MMPL4蛋白和I161在大肠杆菌中表达不明显,而膜外区域O371以包涵体形式高表达。结论 Rv0450c基因原核表达质粒可以成功构建并在大肠杆菌内较好表达,为进一步MMPL4功能研究和抗体制备打下良好基础。

Objective To explore the biological functions of the efflux apparatus with cloning and expression of Rv0450c gene of Mycobacterium turberculosis（M.turberculosis） H37Rv strain.Methods The location and in/out orintation of MMPL4 were analyzed by TMHMM and HMMTOP program.Full length Rv0450c gene and 2 fragments,mmp14-161 and mmp14-371,which encoding an inner（161 amino acids） and an outer membrane region（371 amino acides） of MMPL4 were amplified by PCR from genome of M.tuberculosis H37Rv strain and inserted into prokaryotic expression vector pET28b.The recombinant plasmids were confirmed by double digestion with the enzymes and the DNA sequence were transformed into Escherichia coli BL21（DE3）pLysS strain and induced by isoprogyl B-D-thiogalactopyranoside（IPTG）.The expression of the recombinant protein with 6×histidine（His） tag was identified by sodium dodecyl sulfate-polyacrylamide gel electrophosesis（SDS-PAGE） and was purified through the 6×His affinity chromatography method.Results The size of the constructed recombinant plasmids digested by restricted enzymes of Nhe Ⅰ and Hind Ⅲ was coincident with the expected size.The inserted target gene and its reading frame were coincident with the expression vectors.Expressions of the full legnth MMPL4 and the small inner membrane region were low,while the small outer membrane region was overexpressed in E.coli,but as inclusion bodies.Fruther purification of the intact MMPL4 protein resulted in a purification of a much smaller degradative product.Conclusion The expressions of intact of MMPL4 protein and inner/outer membrane region in E.coli were analyzed,which may facilitate fruther functional study of the Rv0450c gene.