结核分枝杆菌的SirR因子与RelJK毒素-抗毒素蛋白之间的交互作用及其调控细菌生长的分子机制研究

项目名称： 结核分枝杆菌的SirR因子与RelJK毒素-抗毒素蛋白之间的交互作用及其调控细菌生长的分子机制研究

项目编号： No.31301079

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 杨敏

作者单位： 华中农业大学

项目金额： 25万元

中文摘要： 结核分枝杆菌（MTB）具有复杂的细胞生长调控机制如能形成持续性感染是结核病难以根治的重要原因之一，然而目前对MTB生长调控的分子机制还很不清楚。针对这一科学问题，基于实验室已有的工作，本项目拟在系统鉴定转录抑制子SirR与抗毒素蛋白RelJ以及毒素蛋白RelK三者之间交叉相互作用的基础上，进而探讨它们对MTB生长调控的影响。首先，利用Pull-down、表面等离子共振（SPR）、免疫共沉淀等技术系统分析SirR与RelJ、RelK之间的相互作用。然后，采用凝胶阻滞、SPR和报告基因表达等技术剖析蛋白相互作用对relJK操纵子及SirR转录活性的影响。最后，通过构建重组分枝杆菌菌株（基因敲除、超表达以及共表达等）并比较它们的生长速率，阐明交互作用与分枝杆菌细胞生长调控之间的内在联系。本项目研究将有助于发现MTB生长调控新机制和为结核病的防控提供新思路，具有重要的理论价值和潜在实际意义。

中文关键词： 毒素抗毒素系统；SirR；转录调控；蛋白质互作；

英文摘要： Persistence and other complicated growth regulation of Mycobacterium tuberculosis (MTB), has become one of the biggest obstacles in treatment of tuberculosis(TB). However, the involved mechanisms are largely unclear. To address this issue, we will identify the cross-interaction of a repressor of SirR and antitoxin RelJ /toxin RelK basis on previous work ，and further uncover the effect of the interactions on mycobacterial growth. Firstly, we will detect the cross-interaction of SirR and RelJ/K by using the pull-down, surface plasma resonance（SPR）, and immune coprecipitation assays. Secondly, we will analyze the effect of protein interactions on the RelJ/K operon activity as well as the SirR transcription regulation activity by EMSA, SPR and report gene expression assays. Finally, we will illustrate the correlation between cross-interaction and cell growth regulation by constructing recombinant strains (Knockout, Over-expression, and Co-expression) and comparing their growth rate. This study would uncover novel mechanism of regulation of mycobacterial growth, and provide new ideas for the prevention and control of TB.

英文关键词： toxin-antitoxin system；SirR；transcriptional regulation；protein-protein interaction；