结核分枝杆菌Rv1512基因的特异性和克隆表达

Cloning,Prokaryotic Expression and Specificity Identification of Mycobacterium Tuberculosis Rv1512 Gene

目的鉴定结核分枝杆菌Rv1512基因的特异性,克隆表达该基因并获得其重组蛋白。方法设计Rv1512基因的特异性引物,并鉴定其特异性。构建pET30a(+):Rv1512重组质粒,阳性克隆转化入大肠杆菌BL21。经Ni+-NTA层析柱纯化融合蛋白,通过SDS-PAGE鉴定该蛋白及其纯度。结果结核分枝杆菌Rv1512基因的特异性被证实;经测序分析证实Rv1512原核表达质粒构建正确,SDS-PAGE结果显示在40 kD处呈现单一蛋白条带。结论结核分枝杆菌Rv1512基因具有较好的特异性;成功构建表达载体pET30a(+):Rv1512并获得重组蛋白,为辅助诊断结核分枝杆菌的感染奠定基础。

Objective To identification of specific antigens Rv1512 of Mycobacterium tuberculosis.To construct expression vector carrying Mycobacterium tuberculosis（M.tb） Rv1512 gene and express it in E.coli.Methods Design Rv1512 gene-specific primers,and identify its specificity.The positive clones of expression recombinant plasmids pET30a（＋）：Rv1512 were constructed and then transformed into BL21.The fusion protein was purified by Ni＋-NTA column and detected by SDS-PAGE.Results Mycobacterium tuberculosis Rv1512 gene specificity was confirmed.Sequence analysis confirmed that the identification of prokaryotic expression plasmid was correct.The SDS-PAGE results showed that the molecular weight of Rv1512 fusion protein was about 40 kD.Conclusion Mycobacterium tuberculosis Rv1512 gene has better specificity.The expression recombinant plasmids pET30a（＋）：Rv1512 was suecessfully constructed,recombinant Rv1512 protein is produced,which laid the foundation for application in the diagnosis of Mycobacterium tuberculosis.