牛分枝杆菌PE_PGRS62基因克隆及在耻垢分枝杆菌中的表达

Cloning of Mycobacterium bovis PE_PGRS62 gene and its expression in Mycobacterium smegmatis

为构建表达牛分枝杆菌(M.bovis)PE_PGRS62蛋白的重组耻垢分枝杆菌,本研究以M.bovis基因组DNA为模板PCR扩增PE_PGRS62基因,获得大小约为1 515 bp的目的片段,并克隆到大肠杆菌-分枝杆菌穿梭表达载体pMV261中,将重组pMV-PE_PGRS62穿梭表达载体电转化到耻垢分枝杆菌内,42℃热诱导重组耻垢分枝杆菌,通过SDS-PAGE和免疫印迹分析表达产物并鉴定其生物学活性。经SDS-PAGE分析,表达的PE_PGRS62蛋白分子量约为60 ku,免疫印迹结果表明,表达的PE_PGRS62蛋白与兔抗M.bovis阳性血清反应形成一条特异性蛋白条带。本实验为进一步研究M.bovis致病机理奠定了基础。

To construct recombinant Mycobacterium smegmatis expressing PE_PGRS62 protein of Mycobacterium bovis,the PE_PGRS62 gene from a M.bovis strain was amplified by PCR with a pair of primers designed according to the sequence in GenBank.A 1,515 bp gene was amplified and cloned into the shuttle vector pMV261.The recombinant plasmid of pMV-PE_PGRS62 was transformed into M.smegmatis by electroporation and the expression of PE_PGRS62 protein was induced at 42 ℃.SDS-PAGE analysis indicated that the expressed recombinant protein of PE_PGRS62 was about 60 ku.The western blot analysis confirmed the recombinant protein was recognized by positive serum anti-M.bovis.These results provided a foundation for further study on the pathogenesis of M.bovis.