利用基因密码子扩展技术调控结核分枝杆菌功能基因的方法研究及应用

It is an urgent need for the development of new and effective drugs and vaccines for prevention and treatment of tuberculosis. The aim of this study is to use new tools developed from chemical biology methods to study the major infectious pathogens. The genetic code expansion method is utilized to regulate expression of mycobacteria genes of interest. The traditional gene regulation methods, which typically control gene transcription, often have leaky gene expression issues. In contrast, the genetic code expansion technique using orthogonal tRNA/tRNA synthetase pair ensures the tight control of gene expression of Mycobacterium tuberculosis (Mtb) in culture and inside host cells with high fidelity in the protein translation level. This new regulatory system allows us to evaluate and validate candidate drug targets in vitro and in vivo, providing basis for further screening of small molecule inhibitors. In the meantime, we will also use this system to construct non-natural amino acid-dependent and replication-defective recombinant Mtb strains and to demonstrate its feasibility as a new type of vaccine. These results will facilitate further studying the relationship between immunization response period, intensity and protection efficacy in future.

在新型抗结核药物、疫苗亟待开发的背景下，积极拓展新技术用以寻找并开发新型高效的抗结核预防、治疗手段成为一项意义重大、刻不容缓的工作。本课题旨在利用化学生物学研究获得的技术手段来研究重大传染病病原菌的防治方法。将基因密码子扩展技术拓展应用于结核分枝杆菌的遗传调控，借助此技术的严格正交性确保其对基因表达调控的严谨可控性，克服传统转录调控开关工具存在的泄漏表达问题，在蛋白翻译层面上实现对感染宿主体内病原菌的基因表达的严谨调控。基于这一新型调控系统，得以同时在体外、体内环境下对全新药物靶标的可成药性进行评估、确证，为下一步针对性的小分子抑制剂筛选提供可行性依据。同时，我们也将利用这一系统构建非天然氨基酸依赖的复制缺陷性重组结核菌并对其作为新型结核疫苗的可行性进行概念验证研究，为将来进一步研究结核疫苗免疫响应周期、强度与免疫保护效果的关系奠定基础。

基因功能的研究、新靶标的探寻与其可成药性的验证工作离不开高效严谨的遗传调控工具。基于密码子扩展技术的生物正交性及其严谨的可控性，本项目致力于将此技术拓展至结核分枝杆菌中以建立一套新型高效的遗传调控工具，克服传统转录调控开关工具存在的泄漏表达问题，在蛋白翻译层面上实现对感染宿主体内病原菌的基因表达严谨调控。在成功构建这一新型调控系统的基础上，我们也得以同时在体外、体内环境下对新型候选药物靶标进行评估，为下一步针对性的小分子抑制剂药物开发提供可行性依据。同时，我们也利用这一系统构建了非天然氨基酸依赖的复制可控型重组结核杆菌，并对其作为新型结核疫苗及肿瘤免疫治疗的可行性进行概念验证研究，此部分工作也为将来进一步研究结核疫苗免疫响应周期、强度、免疫保护效果及安全性的关系奠定基础。