结核分枝杆菌CYP143A1基因(Rv1785c)敲除菌株的构建

Construction of a CYP143A1 gene(Rv1785c)deletion mutant of Mycobacterium tuberculosis

目的为深入了解和探索结核分枝杆菌CYP143A1基因(Rv1785c)功能,构建针对该基因的敲除菌株。方法采用噬菌体介导的基因敲除技术,设计并采用聚合酶链反应(PCR)扩增待敲除基因Rv1785c左、右臂,与p0004s质粒连接,构建同源重组质粒p0004s-△Rv1785c。再将p0004s-△Rv1785c质粒与phAE159质粒连接,构建phAE159-△Rv1785c噬菌粒。将获得的噬菌粒导入耻垢分枝杆菌mc^(2)155,获得整合有同源交换位点的重组噬菌体,经体外扩增收集高滴度噬菌体,转染结核分枝杆菌H37Rv,筛选阳性克隆并通过PCR法及基因测序验证敲除株构建结果。结果成功构建了包含Rv1785c上下游同源臂的等位交换质粒,并将该质粒与含有分枝杆菌温敏型噬菌体元件的穿梭质粒phAE159连接获得重组噬菌粒,最终经转染进入H37Rv。PCR及基因测序结果表明H37Rv CYP143A1基因敲除菌株构建成功。结论首次成功构建了结核分枝杆菌H37Rv CYP143A1基因敲除菌株,为后续CYP143A1基因功能研究提供了关键材料,奠定了重要的物质基础。

Objective To explore the function of Mycobacterium tuberculosis(Mtb)CYP143A1 gene,a CYP143A1 gene knockout strain was constructed in this study.Methods Phage-mediated gene knockout technology was used to construct a homologous recombination plasmid p0004s-△Rv1785c,which was connected with phAE159 to construct a phAE159-△Rv1785c phagemid.The obtained phagemid was then introduced into Mycobacterium smegmatis mc^(2)155 to construct a recombinant phage integrated with homology exchange sites.The phage was amplified in vitro,and then transfected into Mtb H37Rv.The construction result of the Rv1785c-knockout strain was verified by PCR and gene sequencing.Results An allelic exchange plasmid containing the upstream and downstream homology arms of Rv1785c was successfully constructed,and the plasmid was ligated with the shuttle plasmid phAE159 containing mycobacterial temperature-sensitive phage elements to obtain the recombinant phagemid.Phage was transfected into Mtb H37Rv.PCR and gene sequencing results showed that the Mtb H37Rv CYP143A1 gene knockout strain was successfully constructed.Conclusion The CYP143A1 gene deletion mutant of Mtb H37Rv is successfully constructed for the first time,which provided a very useful tool for further Mtb CYP143A1 gene function research.