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目的 :通过同源重组方法构建粪肠球菌密度感应调控基因luxS基因缺陷突变菌株。方法 :利用PCR扩增粪肠球菌luxS基因的上、下游片段(up、dn)和红霉素抗性基因片段(erm),依次采用相应的双酶切反应,将这些片段连接入载体pUC18,以构建目的质粒Puemrd重组载体。采用同源重组方法,将红霉素耐药基因erm置换粪肠球菌基因组中的luxS基因,并在含抗性标记的选择性培养基中筛选出阳性克隆。结果:经酶切鉴定,目的质粒各片段插入无误,插入片段的测序报告也显示碱基无错配,成功构建粪肠球菌luxS基因缺陷株。结论:本研究成功构建的粪肠球菌luxS基因突变株,为进一步研究该基因在生物膜中的作用及其调控机制提供了技术平台。
OBJECTIVE: To construct the luxS gene-deficient mutants of density-sensitive genes of Enterococcus faecalis by homologous recombination. Methods: The upstream and downstream fragments (up, dn) and erythromycin resistance gene fragment (erm) of luxS gene of Enterococcus faecalis were amplified by PCR. The fragments were ligated into vector pUC18, To construct the purpose plasmid Puemrd recombinant vector. The homologous recombination method was used to replace the luxS gene in the Enterococcus faecalis genome with erm-resistant gene erythromycin, and the positive clones were screened in selective media containing resistance markers. Results: The result of restriction enzyme digestion indicated that the insert of the target plasmid was correct, and the sequencing report of the inserted fragment also showed that there was no mismatch of bases and the luxS gene defective strain of Enterococcus faecalis was successfully constructed. Conclusion: The mutants of luxS gene of Enterococcus faecalis successfully constructed in this study provide a technological platform for further studying the role of this gene in biofilm and its regulatory mechanism.