结核分枝杆菌Rvl884c和Rv0867e基因的克隆表达及其促生长作用的研究

Prokaryotic expression and biological functions of Mycobacterium tuberculosis Rv1884c and Rv0867c genes

目的构建结核分枝杆菌Rv1884c和Rv0867c基因的原核表达质粒，获得结核分枝杆菌Rv1884c和Rv0867c基因的表达蛋白，并初步研究其促生长作用。方法制备结核分枝杆菌基因组DNA，采用PCR技术扩增目的基因片段；将2个片段分别克隆入克隆载体pGEX-4T-1和pUC19，再分别克隆入原核表达载体pGEX-4T-1和pPRO—EXHT，经序列测定证实正确后，再经异丙基硫代-8-D-半乳糖苷（IPTG）诱导表达GST标记的Rv1884c融合蛋白和His标记的Rv0867c融合蛋白；用十二烷基硫酸钠-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Rv1884c和Rv0867c基因，构建了具有正确基因序列的质粒载体pGEX-4T-1-Rv1884c和pPRO—EXHT—Rv0867c，转化人大肠杆菌DH5a中经诱导产生高水平的表达产物。经SDS分析，在相对分子质量为45000和80000处出现新生蛋白带，凝胶薄层扫描检测表达量分别约占菌体蛋白的18．3％和23．7％。用GSTrapFF亲和层析柱和Ni^2＋-NTA纯化柱进行蛋白纯化，并研究这两种蛋白对藤黄微球菌、BCG和结核分枝杆菌H37Rv的促生长作用。结论成功克隆了结核分枝杆菌Rv1884c和Rv0867c基因并得到了其大肠杆菌表达产物，为进一步研究Rvl884c和Rv0867c基因蛋白的活性及其功能，以及研究结核分枝杆菌快速促生长作用奠定了基础。

Objective To construct fusion gene and prokaryotic expression plasmid encoding the Rv1884e and Rv0867c genes in Mycobacterium tuberculosis （M. tb）. The fusion protein was expressed efficiently in E. eoli cells. Methods The Rv1884c and Rv0867c genes were amplified by polymerase chain reactions （PCR） with specific primers from genomic DNA of M. tb H37Rv strain, and cloned into pGEX-4T- land pUC19 vectors. Rv1884c and Rv0867e were subeloned into the expression vector pGEX-4T-1 and pPRO-EXHT followed by DNA sequencing. The plasmids were transformed into E. coli DH5α and induced to produce GST-fused Rv1884e and His-fused Rv0867c fusion protein. The protein molecular weight and expression format was analyzed by SDS-PAGE. Results The recombinant expressive vectors pGEX-4T-1- Rv1884e and pPRO-EXHT-Rv0867c were constructed. The DH5α strains of E. call with recombinant plasmid showed high level of Rv1884c and Rv0867c gene expressions after IPTG induction. The SDS-PAGE showed that the plasmids expressed Rv1884c and Rv0867c fusion proteins with molecule weight of 45 000 and 80 000. The recombinant protein accounted for 18.3% and 23.7% of total bacteria protein. The expressed proteins could be purified via GSTrap FF and Ni^2＋ -NTA system kits in denatured condition. Conclusions Mycobacterium tuberculosis Rv1884c and Rv0867c genes have been cloned and expressed Successfully in E. coli DH5α. The results lay a basis for further study of fast cultivation in Mycobacterium tuberculosis and investigation of their activities and functions.