结核杆菌基因组RD1区毒力蛋白分泌相关基因Rv3871的克隆、表达及生物信息学分析

Cloning, expression and bioinformatic analysis of Rv3871 gene related to Mycobacterium tuberculosis virulent protein secretion

目的对结核分枝杆菌H37Rv毒株RD1毒力分泌系统Rv3871基因进行克隆和蛋白表达,运用生物信息学分析该基因在结核杆菌毒力蛋白分泌过程中的作用。方法以结核分枝杆菌H37Rv基因组为模板,以PCR扩增出完整的Rv3871基因,将其插入原核表达载体pET32a(+),对其核苷酸序列进行测定。利用生物信息学软件对结核杆菌Rv3871进行结构功能分析并与多种分枝杆菌进行同源性比对。结果经分子克隆的Rv3871酶切片段与预期大小符合,基因序列与Genbank报道一致,SDS-PAGE检测到相对分子质量为84000的特异性表达蛋白,生物信息学分析发现两个FtsK/SpoEⅢ样结构域,同源性比对则发现在致病性分枝杆菌与卡介苗等非致病菌Rv3971基因对应区域的结构完整程度有较为显著的差别。结论本研究通过对结核分枝杆菌RV3871基因进行分子克隆,特异蛋白表达和基因序列测定,提供了该基因的结构功能特征,并通过生物信息学与同源性对比分析,发现Rv3871基因在致病和非致病分枝杆菌毒力分泌作用中的结构和功能差异,为结核病发病机制阐明及其治疗药靶的筛选提供理论和实验依据。

Objective To clone and express the Rv3871 gene related to the virulent protein secretion of Mycobacterium tuberculosis and analyze its molecular structure, function and homology using bioinformatic approach. Methods A pair of primers was designed to amplify the Rv3871 gene, which was subcloned into the prokaryotic plasmid pET32a （＋）. The recombinant plasmid was identified by sequence analysis and the expressed recombinant protein by SDS-PAGE. The structure, function and homology alignment of Rv3871 were analyzed comparatively against other mycobacteria. Results The restriction fragments through molecular cloning matched perfectly in size with our prediction. The gene sequence was consistent with the corresponding sequence in GenBank. The expression protein was detected by SDS-PAGE with a molecular weight of 84 kD. Two FtsK/SpoE Ⅲ domains were found by bioinformatic analysis. The homology results showed distinct differences between Rv3871 of the pathogenic M. tuberculosis and its counterparts in non-pathogenic mycobacteria. Conclusions Molecular cloning, expression and sequencing identify the structural and functional characteristics of Rv3871. The structural and functional differences of the gene between pathogenic and non-pathogenic mycobacteria identified by bioinformatics provide some evidence for the pathogenesis and drug targets of tuberculosis.