结核分枝杆菌Rv0867c基因的克隆表达及其纯化

目的构建结核分枝杆菌Rv0867c基因的原核表达质粒,获得结核分枝杆菌Rv0867c基因的表达蛋白。方法制备结核分枝杆菌基因组DNA,采用聚合酶链反应(PCR)技术扩增目的基因片段;通过克隆载体pUC19构建质粒载体pUC19-Rv0867c,经序列测定证实正确,双酶切后连接于表达载体pPRO-EXHT,转化入大肠杆菌DH5α中,再经IPTG诱导表达带His标签的Rv0867c融合蛋白;用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的相对分子质量大小及表达形式。结果成功扩增出了结核分枝杆菌Rv0867c基因,构建了具有正确基因序列的表达载体pPRO-EXHT-Rv0867c,转化入大肠杆菌DH5α中,经诱导产生高水平的表达产物。经SDS分析,在80 kD处出现新生蛋白带,凝胶薄层扫描检测表达量约占菌体蛋白的23.7%。该融合蛋白以包涵体的形式存在,用Ni²⁺-NTA纯化柱在变性条件下进行纯化。结论成功克隆了结核分枝杆菌Rv0867c基因并得到了其大肠杆菌表达产物,为进一步研究Rv0867c基因蛋白的活性及其功能,以及结核分枝杆菌快速促生长作用奠定了基础。

Cloning, prokaryotic expression and purification of Mycobacterium tuberculosis Rv0867c gene

Objective To construct prokaryotic expression plasmid encoding Rv0867C gene from Mycobacterium tuberculosis(M.tb),to express efficiently the fusion proteins in E.coli. Methods The Rv0867c gene was amplified by polymerase chain reactions(PCR) with specific primers from genomic DNA of M.tb H37Rv strain,and cloned into plasmid pUC19.Then Rv0867C was subcloned into the expression vector pPRO-EXHT.The plasmid pPRO-EXHT-Rv0867c was transformed into E.coli DH5α and induced by IPTG.The Rv0867c fusion protein expressed was analyzed by SDS-PAGE.Recombinant His-6-fused protein was purified by Ni2+-NTA purification system. Results The length of PCR products was 1224bp.The result of DNA sequencing showed that the amplified Rv0867C gene was exactly consistent with the sequence reported in Genebank.The recombinant expressive plasmid pPRO-EXHT-Rv0867c was constructed.The E.coli DH5α strain with recombinant plasmid showed high level of Rv0867c gene expression after IPTG induction.The plasmid pPRO-EXHT-Rv0867c expressed a Rv0867c fusion protein with relative molecule mass 80,000KDa.The protein band amounted to 23.7% of total bacteria protein,SDS-PAGE analysis showed that the fusion protein mainly existed in inclusion body.The expressed protein could be purified via Ni²⁺-NTA system kit in denatured condition. Conclusion Mycobacterium tuberculosis Rv0867c gene has been cloned and expressed successfully in E.coli DH5α.The Results lay a basis for further study of fast cultivation for Mycobacterium tuberculosis.