结核分枝杆菌WhiB2蛋白的表达纯化及理化性质和生物信息学分析

Expression and purification of Mycobacterium tuberculosis WhiB2 and analysis of its physicochemical properties and structure based on bioinformatics

目的探索结核分枝杆菌(Mycobacterium tuberculosis,Mtb)WhiB2蛋白的作用机制。方法构建Mtb中pET28a-WhiB2重组表达载体,异源表达并纯化WhiB2蛋白和包涵体WhiB2蛋白;圆二色谱和核磁共振分析包涵体WhiB2与非变性WhiB2蛋白的差异;加入Fe^(2+)恢复WhiB2蛋白的铁硫簇;核磁谱图分析WhiB2与WhiBMtb基因上游的启动子序列相互作用;生物信息学分析其三级结构、三级结构比对和相互作用蛋白质。结果复性的WhiB2与非变性WhiB2的结构基本一致,在破坏了铁硫簇的WhiB2中加入Fe^(2+)能够成功恢复自身的铁硫簇,WhiB2可以与WhiBMtb/WhiB2Ms基因上游的启动子序列结合。结论Mtb WhiB2蛋白在结核病发病机制中起关键作用,为进一步探索抗Mtb的新型靶点提供基础资料。

ObjectiveTo investigate the mechanism of Mycobacterium tuberculosis(Mtb)WhiB2 in the pathogenesis of tuberculosis.MethodsA recombinant vector of pET28a-WhiB2 for heterogeneous expression of WhiB2 was constructed.The target protein WhiB2 and the inclusion bodies were purified.The differences between denatured and non-denatured WhiB2 were analyzed by circular dichroism and nuclear magnetic resonance.Ferrous ion(Fe^(2+))was used to restore the iron-sulfur cluster of WhiB2.The interaction between WhiB2 and the upstream promoter sequence of the WhiBMtb gene was analyzed by nuclear magnetic resonance.The tertiary structure of WhiB2 and interacting proteins were analyzed and protein structure alignment was performed based on bioinformatics.ResultsThe structure of the renatured WhiB2 was basically the same as that of the non-denatuous WhiB2.In addition,Fe^(2+)could restore the iron-sulfur cluster of WhiB2.It was found that WhiB2 could bind to the upstream promoter sequence of the WhiBMtb/WhiB2Ms gene.ConclusionsMtb WhiB2 played a key role in the pathogenesis of tuberculosis,which would contribute to future exploration of novel targets against Mtb.