粪肠球菌luxS基因缺陷菌株的构建

上海口腔医学 ›› 2015, Vol. 24 ›› Issue (4): 410-414.

粪肠球菌luxS基因缺陷菌株的构建

何智妍¹, 程岚², 王玉霞¹, 黄正蔚¹

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,2.牙周病科,上海市口腔医学重点实验室,上海 200011

收稿日期:2014-08-12 出版日期:2015-08-20 发布日期:2015-09-10
通讯作者: 黄正蔚,Tel: 021-63135412,E-mail: huang_zhengwei@hotmail.com E-mail:huang_zhengwei@hotmail.com
作者简介:何智妍（1984-）,女,技师,E-mail: zyhe23@126.com
基金资助:
国家自然科学基金(81300866,81371143)

Construction of luxS gene knockout mutant of Enterococcus faecalis

HE Zhi-yan¹, CHENG Lan², WANG Yu-xia¹, HUANG Zheng-wei¹

1.Department of Endodontics, 2.Department of Periodontology,Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Received:2014-08-12 Online:2015-08-20 Published:2015-09-10
Supported by:
Supported by National Natural Science Foundation of China (81300866 and 81371143)

摘要/Abstract

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。

关键词: 粪肠球菌, 生物膜, luxS基因

Abstract: 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 pUC18 by corresponding double digests. After allelic exchange, the luxS knockout mutants strains were selected on 30 μg/mL 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 faecalisluxS gene has been successfully disrupted with homologous recombination. This mutant strain sets a good foundation for further functional study.

Key words: Enterococcus faecalis, Biofilm, luxS geneShanghai J Stomatol, 2015, 24(4):410-414.

中图分类号:

R780.2

何智妍, 程岚, 王玉霞, 黄正蔚. 粪肠球菌luxS基因缺陷菌株的构建[J]. 上海口腔医学, 2015, 24(4): 410-414.

HE Zhi-yan, CHENG Lan, WANG Yu-xia, HUANG Zheng-wei. Construction of luxS gene knockout mutant of Enterococcus faecalis[J]. Shanghai Journal of Stomatology, 2015, 24(4): 410-414.

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