分枝杆菌噬菌体重组系统及其应用

Recombineering Based on Mycobacteriophage and Its Application

噬菌体是微生物遗传学研究的有力工具及源泉。分枝杆菌噬菌体也是构建分枝杆菌,尤其是结核分枝杆菌遗传研究工具的基础。目前,基于分枝杆菌噬菌体重组酶的重组系统是国际热点。总结了近年来基于分枝杆菌噬菌体Che9c重组酶gp60、gp61所构建的分枝杆菌重组工程体系及其在分枝杆菌基因组研究方面的应用,并结合实验室工作展望了其研究前景。该体系不依赖细菌自身的RecA系统,不需要限制性内切核酸酶和DNA连接酶,不需要复杂的体外操作,只需表达分枝杆菌噬菌体重组酶,从而使结核分枝杆菌基因敲除、基因敲入及点突变和构建分枝杆菌噬菌体突变株更方便。这为分枝杆菌及其噬菌体基因诱变及基因功能研究提供了迅捷的新途径。

Bacteriophage is a powerful tool to address fundamental genetics issues. This is true for Mycobacteriophages too, a well-documented resource for Mycobacterium tuberculosis genetics. Recent developments of mycobacterial recombineering system, which is based on mycobacteriophage Che9c-encoded proteins, are reviewed and its application in basic M. tuberculosis biology is outlined. The advantage of this system is that it is independent of bacterial recA system, restriction endonuclease and DNA ligase, and complex in vitro manipulation. The expression of Che9c-encoded exonuclease and recombinase could substantially complete the construction of gene knockouts or knock-ins, point mutants and mycobacteriophage mutants. The mycobacterial recombineering system is a facile new tool to study gene function and for mutation analysis.