结核分枝杆菌Rv3425与Rv3614c的相互作用

Protein-protein interaction between Rv3425 and Rv3614c in Mycobacterium tuberculosis

为探究结核分枝杆菌脯氨酸-脯氨酸-谷氨酸(PPE)蛋白家族Rv3425的生物学功能,利用免疫共沉淀联合质谱分析,在卡介苗(BCG)中筛选与Rv3425相互作用的蛋白。首先,以聚合酶链反应(PCR)扩增获得Rv3425基因,并克隆于pMV261载体。将重组载体转化入BCG,裂解细胞,蛋白免疫印迹法证实目的蛋白可在BCG中表达。通过免疫共沉淀,用特异抗体分离出BCG中的Rv3425蛋白复合体,质谱鉴定其成分,在美国国立生物技术信息中心(NCBI)数据库中检索各蛋白的功能,筛选与Rv3425相互作用的蛋白,并用谷胱甘肽S-转移酶(GST)pulldown验证。结果显示,免疫共沉淀联合质谱分析筛选到10个与Rv3425相互作用的蛋白,在细胞内Rv3425协同作用下参与肽聚糖合成途径、分枝菌酸合成途径、ESX-1蛋白分泌系统及细菌毒力调控。GST pulldown验证发现Rv3614c与Rv3425可体外结合。本研究证实Rv3425与Rv3614c存在相互作用,Rv3425可能与ESX-1蛋白分泌系统及结核分枝杆菌致病机制密切相关。

This study aims to investigate the function of Rv3425 in Mycobacterium tuberculosis （M. tuberculosis） by screening its interacting proteins in bacillus Calmette-Gu6rin （BCG） via coimmunoprecipitation coupled with mass spectrometry. Regions coding for Rv3425 were amplified by polymerase chain reaction （PCR） and cloned into the pMV261 vector. The recombinant vector was transformed into BCG, and the expression of the fusion protein was verified by Western blotting analysis. The Rv3425 protein complex formed in BCG （rBCG） were obtained by coimmunoprecipitation combined with anti-His specific antibody. Mass spectrometry was used to identify protein components in the complex; the function of each protein was obtained from the National Center for Biotechnology Information （NCBI）. The candidate proteins interacting with Rv3425 were verified by glutathione S-transferase （GST） pulldown assay. Ten proteins interacted with Rv3425 were obtained. Rv3425 could coordinate with a variety of proteins implicated in intermediary metabolism and respiration, cell wall and cell processes as well as latent infections. The results also demonstrated that Rv3614c protein implicated in ESX-1 secretion system and possible pathogenic mechanism could bind to Rv3425 in vitro.