基于分子对接技术的结核分枝杆菌耐异烟肼和利福平的分子机制研究

项目名称： 基于分子对接技术的结核分枝杆菌耐异烟肼和利福平的分子机制研究

项目编号： No.81460315

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 袁小亮

作者单位： 赣南医学院

项目金额： 46万元

中文摘要： 靶基因位点突变是结核分枝杆菌（MTB）产生耐药的主要原因。前期研究发现 MTB 5个KatG 和6个rpoB基因新突变位点(J Clin Microb.2012,7)。这些位点与MTB对异烟肼（INH）和利福平（RFP）耐药关系及其分子机制尚不清楚。本项目拟用分子克隆、分子对接、DNA微阵列等技术，进一步研究①野生型和突变型KatG蛋白的功能差异；②耻垢分枝杆菌和M.tuberculosis H37Ra导入重组突变型KatG和rpoB基因穿梭质粒的MIC变化；③野生型和诱变型KatG和rpoB蛋白与INH和RFP相互作用的差别；④耐多药株在药物诱导作用下的外排泵基因表达及活性的变化。本研究旨在明确上述突变位点与MTB对INH和RFP耐药的相关性，阐明相对应的KatG和rpoB变异蛋白引起MTB耐INH和RFP的分子机制。为结核病耐药检测基因芯片的开发和药物新靶点的发现提供理论和实验依据。

中文关键词： 结核分枝杆菌；耐药性；基因突变；分子对接

英文摘要： Ponit mutation in the targeted gene is the main mechanism of drug resistance in Mycobacterium tuberculosis（MTB）. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro and Asp695Gly), and six new allelesin the rpoB gene(Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu and Lys972Thr), were identified in our previous study(J Clin Microb.2012,7).However, it remains to be ascertained if these mutations are associated with INH or RFP resistance in Mycobacterium tuberculosis.To investigate the association between these mutations and INH or RFP resistance in Mycobacterium tuberculosis, the following studies will be performed: ①cloning wild katG gene and 5 novel katG mutants, and comparing their INH oxidase, peroxidase, and catalase activities; ②transforming the mutated rpoB and katG gene into wild-type Mycobacterium smegmatis and M.tuberculosis strain H37Ra,and determining the INH or RFP MIC（minimum inhibitory concentration）change of the recombinant Mycobacterium smegmatis and M.tuberculosis strain H37Ra;③ constructing the models of wild-type and mutated rpoB(katG) protein using the Protein Fold Recognition Server, docking wild-type and mutated rpoB (katG) protein with rifampin (isoniazid) using AutoDock 4.0, and comparing the molecular dynamics and conformational changes of the mutant rpoB-rifampin (KatG-isoniazid) complex with the wild-type rpoB-rifampin (KatG-isoniazid) complex. Moreover, to investigate the role of efflux pumps in MTB drug resistance, we will construct DNA microarrays containing probable drug efflux pump genes of MTB, and detect changes in the expression of these genes of the multidrug-resistant Mycobacterium tuberculosis clinical isolates on exposure of INH or RFP. The aim of this study is to explain the molecular mechanism of INH or RFP resistances conferred by the mutated katG or rpoB proteins, and provide useful information for discovering novel drug target points and developing the gene chips to detect drug resistance in Mycobacterium tuberculosis.

英文关键词： Mycobacterium tuberculosis;drug resistance;gene mutation;molecular docking