结核分枝杆菌同源重组基因敲除系统的构建和应用

Development and application of homologous recombination knockout system in Mycobacterium tuberculosis

【目的】结核分枝杆菌同源重组效率很低,突变株的构建需要半年之久。本研究的目的在于构建一种用于在结核分枝杆菌中进行基因快速敲除、且易于筛选的高效同源重组系统。【方法】野生型结核分枝杆菌转化含有SacB反向选择标记、且能诱导表达两种同源重组酶gp60和gp61的质粒pSL002。然后分别将靶基因的两个同源臂克隆入到含有hyg(潮霉素)抗性基因和gfp(绿色荧光蛋白)基因的重组质粒pSL001中,再将靶基因同源臂-loxP-hyg-gfp-loxP片段从pSL001切下,转化含有pSL002的野生型结核分枝杆菌,一步得到双交换突变株。再将含有SacB反向选择标记、且表达Cre重组酶的质粒pSL003转化入结核分枝杆菌双交换突变株中,切除两个loxP之间的hyg抗性基因和gfp基因,得到无痕缺失突变株。最后利用含有2%蔗糖的琼脂糖平板去除含有SacB反向选择标记的质粒pSL002和pSL003。【结果】在结核分枝杆菌中成功构建了高效同源重组系统,利用该系统构建了rv1364c、pstP跨膜区、pstP胞外区三个突变株,得到双交换突变株的效率为25%-62.5%,从双交换突变株得到无痕缺失突变株的效率为100%。通过gfp作为荧光标记基因,利用NightSea BlueStar蓝光手电筒和滤光眼镜,可以对平板上的基因缺失株直接进行快速判定。【结论】该同源重组系统利用gp60和gp61重组酶,在时间上将在结核分枝杆菌中无痕缺失突变株的构建从6个月缩短到3个月。这是目前为止在结核分枝杆菌中构建突变株最快且效率最高的方法,为加速分枝杆菌功能基因组的研究提供了新的遗传工具。

[Objective] We developed a homologous recombination system to make marker-free mutants in Mtb in an easy screening way.[Methods] The plasmid pSL002 harboring recE and recT-like protein gp60 and gp61 was transformed into wt Mtb in order to increase the recombing efficiency.The two homologous arms of target gene were amplified and cloned into pSL001.The purified homologous arms-loxP-gfp-hyg-loxP fragments were further transformed into Mtb（with pSL002 inside） competent cells to obtain the double cross over（DCO） mutants.The plasmid pSL003 was transformed into DCO competent cells to excise the two loxP sites flanked region.The plasmid pSL002 and pSL003 were removed via the counter selection marker sacB.[Results] An efficient homologous recombination system was developed.Three different marker free mutants： Rv1364c phosphatase domain,PstP extracellular domain and PstP transmembrane domain were created by the system developed in this study.The efficiency to obtain DCO was varying from 25% to 62.5%,while the efficiency from DCO to KO was close to 100%.The gfp reporter was used to screen SCO,DCO and KO.[Conclusion] The homologous system shortens the complicated screening process to 3 month in Mtb.This is the fastest allelic exchange system reported so far,providing novel deletion strategy to the repertoire of mycobacterial genetic tools for constructing unmarked mutations.