结核分枝杆菌Rv1168c蛋白的基因表达、纯化及结构分析

Expression, purification, and characterization of Mycobacterium tuberculosis Rv1168c

摘要:

摘要：【目的】应用原核表达体系对结核分枝杆菌PPE蛋白家族Rv1168c进行高效表达，进一步进行蛋白纯化和结构分析。【方法】以结核分枝杆菌H37Rv基因组为模板，扩增Rv1168c基因，构建pET32a-Rv1168c重组质粒；转化重组质粒到大肠杆菌DH5α并在BL21(DE3)诱导表达，通过十二烷基硫酸钠-聚丙烯酰胺电泳（SDS-PAGE）鉴定Rv1168c在大肠杆菌中的表达情况；Ni-NTA His﹡Bind Resin纯化重组蛋白Rv1168c；SDS-PAGE和质谱分析测定相对分子量后，用圆二色光

Abstract:

Abstract：[Objective] To express and purify the Pro–Pro–Glu (PPE) family protein Rv1168c of Mycobacterium tuberculosis in E. coli. and to study the structure of Rv1168c. [Methods] The Rv1168c gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain genomic DNA and cloned into a prokaryotic expression vector pET32a The resulting recombinant expression plasmid pET32a-Rv1168c was then transformed into the E .coli strain DH5α and a high-level expression E. coli BL21(DE3) was established after induction with Isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE and mass spectrum analysis determined the relative molecular weight of this recombinant Rv1168c protein. It’s secondary and 3D structures were determined by circular dichroism and homologous modeling. [Results] The Mycobacterium tuberculosis Rv1168c gene (971bp) and high purified recombinant Rv1168c protein was obtained. The relative molecular weight of recombinant Rv1168c protein was determined to be 51.5kDa (vector included). Secondary structure of Rv1168c had about 34.4% α helix, 33.7%, β tune, 31.9% random coil at 25℃. Homologous modeling shows Rv1168c as （β/α）5 protein. [Conclusion] This study obtained purified recombinant Rv1168c protein and laid the foundation for exploration of the relationship between the structure and function of Rv1168c in the tuberculosis.