粪肠球菌luxS基因缺陷菌株的构建  

Construction of luxS gene knockout mutant of Enterococcus faecalis

作　　者：何智妍[1] 程岚[2] 王玉霞[1] 黄正蔚[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院·口腔医学院牙体牙髓病科上海市口腔医学重点实验室,上海200011 [2]上海交通大学医学院附属第九人民医院·口腔医学院牙周病科上海市口腔医学重点实验室,上海200011

出　　处：《上海口腔医学》2015年第4期410-414,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81300866,81371143)~~

摘　　要：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。PUPPOSE： To construct quorum sensing luxS knockout mutants of Enterococcus faecalis through homologous recombination. METHODS： The upstream and downstream flank DNA fragments of E. faecalis luxS gene（up, dn） and erythromycin resistance gene（erm） were amplified by PCR. In order to construct recombination plasmid Puemrd, these DNA fragments were inserted into the plasmid p UC18 by corresponding double digests. After allelic exchange, the luxS knockout mutants strains were selected on 30 μg/m L erythromycin plates. RESULTS： With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Puemrd, was constructed correctly. The luxS knockout mutants strains were confirmed by PCR. CONCLUSIONS： Enterococcus faecalis luxS gene has been successfully disrupted with homologous recombination. This mutant strain sets a good foundation for further functional study.

关 键 词：粪肠球菌 生物膜 LUX S基因 

分 类 号：R780.2[医药卫生—口腔医学]