一种高效的葡萄球菌基因敲除方法的建立与应用

Establishment and application of a rapid and efficient method for gene replacement in Staphylococci

目的建立一种高效构建葡萄球菌基因敲除突变株的方法。方法通过融合PCR将vraSR操纵子上、下游同源片段连接起来;通过基于位点特异性重组的Gateway克隆技术将融合PCR产物连接入温度敏感型穿梭质粒pKOR1,转化修饰缺陷型大肠杆菌DC10B,获得同源重组质粒pKOR1-vraSR;电转化表皮葡萄球菌RP62A株;在高温和氯霉素双重选择压力下,质粒通过第1次同源重组整合到表皮葡萄球菌染色体上;转移到无抗生素的培养基中继续培养,发生第2次同源重组;将菌液涂布于含1μg/mL脱水四环素的培养基上进行反向筛选,那些未发生重组的整合菌株因表达必需基因secY的反义RNA而无法生存,最后,挑取菌落通过PCR进行鉴定。结果成功构建了难以转化的表皮葡萄球菌RP62A株的vraSR基因敲除突变株。结论利用大肠杆菌DC10B和穿梭质粒pKOR1对葡萄球菌进行基因敲除的方法简便高效,适用的范围更广泛。

We established a rapid and efficient method for creating knockout mutants in Staphylococci.The upstream and downstream homologous regions of the vraSRoperon were combined by fusion PCR,ligated into temperature-sensitive shuttle plasmid pKOR1 by the Gateway cloning technology,and then transformed into a restriction-defective E.coli strain DC10 B,yielding the recombinant plasmid pKOR1-vraSR.The plasmid was then electroporated into Staphylococcus epidermidis RP62 A strain.Under the selective pressure of high temperature and chloramphenicol,the plasmid was firstly integrated into the chromosome of S.epidermidis through a single crossover event.The integrant strain was then subcultured,and the second crossover occurred subsequently in the absence of antibiotic selection,resulting in either wild-type strain or the mutant,which depended upon where the recombination event occurred.Finally,the culture was plated on the medium containing 1μg/mL anhydrotetracycline for counter selection,several colonies were picked up and analyzed by PCR to identify the mutant.Result showed that a vraSRknockout mutant of the untransformable S.epidermidis RP62 Awas successfully generated in this study.In conclusion,the method for gene replacement in Staphylococci by using E.coli DC10 Band shuttle plasmid pKOR1 is simple and efficient.