关键词: Rv0867c, 分枝杆菌,结核, 基因表达, 聚合酶链反应, 快速培养

Abstract: 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.

Key words: Rv0867c, Mycobacterium tuberculosis, Gene expression, PCR, Fast cultivation