An Inducible Expression System for M. tuberculosis

结核分枝杆菌的诱导表达系统

• 批准号: 7083577
• 负责人: ROBERT N HUSSON
• 金额: $ 8.25万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7083577
• 关键词: Mycobacterium tuberculosis; Nocardiaceae; bacterial genetics; bacterial proteins; biomarker; biotechnology; plasmids; recombinant proteins; transfection /expression vector

DESCRIPTION (provided by applicant): Tuberculosis remains the leading cause of mortality among persons with HIV/AIDS. New approaches to therapy are needed to shorten the duration of therapy, combat drug resistance and kill latent bacteria. Many important advances in understanding the molecular pathogenesis of tuberculosis have been made in the past decade. A major factor in this progress has been the development of molecular genetic tools that allow investigators to manipulate Mycobacterium tuberculosis. While a system for regulated gene expression in mycobacteria was developed several years ago, its relatively high level of basal expression limits its use in many experiments. Defining and characterizing essential genes is a key aspect of defining novel drug targets in M. tuberculosis. The goal of this RO3 small grant application is to adapt an inducible expression system, the positively regulated nitA promoter of Rhodococcus rhodochrous that has minimal basal expression in the absence of induction, for use in mycobacteria. This goal is addressed through Aim 1, in which a) the components of this system will be transferred to a series of replicating and integrating mycobacterial vectors utilizing several selectable markers, b) un-induced expression from this promoter in mycobacteria will be analyzed and the system modified, if needed, to minimize basal expression, and c) the dose response of expression to inducer concentration will be determined. This system will then be used in sub-aim d) to determine whether a gene of interest, such as a potential drug target, is essential. This system will also be tested in sub-aim e) to determine whether it is useful for the expression of full length soluble recombinant mycobacterial proteins in a mycobacterial host, e.g. in M. smegmatis.

描述（由申请人提供）：结核病仍然是艾滋病毒/艾滋病患者死亡的主要原因。需要新的治疗方法来缩短治疗时间、对抗耐药性并杀死潜伏细菌。过去十年，在了解结核病的分子发病机制方面取得了许多重要进展。这一进展的一个主要因素是分子遗传工具的开发，使研究人员能够操纵结核分枝杆菌。虽然分枝杆菌基因表达调控系统几年前就已开发出来，但其相对较高的基础表达水平限制了其在许多实验中的使用。定义和表征必需基因是定义结核分枝杆菌新药物靶点的一个关键方面。 RO3 小额资助申请的目标是调整诱导型表达系统，即红红球菌的正向调节 nitA 启动子，在没有诱导的情况下具有最小的基础表达，用于分枝杆菌。这一目标是通过目标 1 实现的，其中 a) 该系统的组件将利用几个选择标记转移到一系列复制和整合的分枝杆菌载体中，b) 将分析分枝杆菌中该启动子的非诱导表达，并分析如果需要，修改系统以最小化基础表达，并且c)将确定表达对诱导剂浓度的剂量反应。然后，该系统将用于子目标 d) 以确定感兴趣的基因（例如潜在的药物靶标）是否是必需的。该系统也将在子目标 e) 中进行测试，以确定其是否可用于在分枝杆菌宿主（例如分枝杆菌宿主）中表达全长可溶性重组分枝杆菌蛋白。耻垢分枝杆菌。