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.

参考文献

[1] Silveira LF, Baca P, Arias-Moliz MT, et al. Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm [J]. Int J Oral Sci, 2013, 5(3): 146-149.
[2] María Ferrer-Luque C, Teresa Arias-Moliz M, Ruíz-Linares M, et al. Residual activity of cetrimide and chlorhexidine on Enterococcus faecalis -infected root canals[J]. Int J Oral Sci, 2014, 6(1): 46-49.
[3] Ran S, He Z, Liang J. Survival of Enterococcus faecalis during alkaline stress: changes in morphology, ultrastructure, physiochemical properties of the cell wall and specific gene transcripts [J]. Arch Oral Biol, 2013, 58(11): 1667-1676.
[4] Learman DR, Yi H, Brown SD, et al. Involvement of Shewanella oneidensis MR-1 LuxS in biofilm development and sulfur metabolism [J]. Appl Environ Microbiol, 2009, 75(5): 1301-1307.
[5] He Z, Wang Q, Hu Y, et al. Use of the quorum sensing inhibitorfuranone C-30 to interfere with biofilm formation by Streptococcus mutans and its luxS mutant strain[J]. Int J Antimicrob Agents, 2012, 40(1): 30-35.
[6] Shao C, Shang W, Yang Z, et al. LuxS -dependent AI-2 regulates versatile functions in Enterococcus faecalis V583 [J]. J Proteome Res, 2012, 11(9): 4465-4475.
[7] Cai Y, Strømme M, Melhus A, et al. Photocatalytic inactivation of biofilms on bioactive dental adhesives [J]. J Biomed Mater Res B Appl Biomater, 2014, 102(1): 62-67.
[8] Lowery CA, Abe T, Park J, et al. Revisiting AI-2 quorum sensing inhibitors: direct comparison of alkyl-DPD analogues and a natural product fimbrolide[J]. J Am Chem Soc, 2009, 131(43): 15584-15585.
[9] Kolenbrander PE, Palmer RJ, Periasamy S, et al. Oral multispecies biofilm development and the key role of cell-cell distance [J]. Nat Rev Microbiol, 2010, 8(7): 471-480.
[10] 童忠春,倪龙兴,马丽芳, 等. 变异链球菌 luxS 基因对牙菌斑生物膜形成的影响研究[J]. 华西口腔医学杂志, 2009, 27(4): 386-389.
[11] Huang Z, Meric G, Liu Z, et al. LuxS -based quorum-sensing signaling affects Biofilm formation in Streptococcus mutans [J]. J Mol Microbiol Biotechnol, 2009,17(1): 12-19.

[1]	单晓阳, 孙莉青, 王月月, 杨楠, 孙慧斌. 微创开髓下3种根管消毒方法对粪肠球菌清除效果比较[J]. 上海口腔医学, 2022, 31(1): 17-23.
[2]	郑沛, 张羽, 汪飒. 茶黄素对变异链球菌浮游细菌和生物膜的体外抑制作用[J]. 上海口腔医学, 2021, 30(1): 33-37.
[3]	马婧, 李继扬, 杨巧珍, 白昱, 张程, 孙红英. 不同功率密度的姜黄素-光动力疗法对白色念珠菌生物膜的影响[J]. 上海口腔医学, 2020, 29(5): 456-461.
[4]	马健, 邵强, 于毅. 不同龈下楔状缺损充填材料对牙龈卟啉单胞菌生物膜形成的影响[J]. 上海口腔医学, 2020, 29(4): 375-379.
[5]	程刚, 王留宏, 杨惠, 娄新田, 王一霖. 氯己定联合机械清创对种植体周围炎的治疗效果及对患者SF-36评分的影响[J]. 上海口腔医学, 2020, 29(4): 400-404.
[6]	陆佳萍, 黄正蔚, 钱文昊. 根管负压冲洗对根尖推出物及根管内粪肠球菌的影响[J]. 上海口腔医学, 2019, 28(6): 601-604.
[7]	汪嘉, 赵申, 冉淑君, 孙喆, 梁景平. 不同根管冲洗方式对粪肠球菌生物膜清除效果评价[J]. 上海口腔医学, 2019, 28(3): 246-250.
[8]	何智妍, 周薇, 黄正蔚, 梁景平, 姜葳. 变形链球菌luxS基因突变对口腔混合细菌生物膜的影响[J]. 上海口腔医学, 2019, 28(2): 113-117.
[9]	孙楚文, 朱亚琴. 不同根管消毒方法对粪肠球菌清除作用的体外研究[J]. 上海口腔医学, 2017, 26(2): 134-138.
[10]	张富华，李矛，韦智君，赵兵. 纳米银颗粒联合氢氧化钙对饥饿期粪肠球菌生物膜的作用[J]. 上海口腔医学, 2016, 25(1): 11-15.
[11]	张冬梅，刘静波，潘亚萍，潘佳雨，徐秋芳. 戈登链球菌对牙龈卟啉单胞菌生物膜形成的影响[J]. 上海口腔医学, 2015, 24(6): 641-644.
[12]	许雪松，朱亚琴. 根管预备尺寸对Er:YAG激光消毒效果的影响[J]. 上海口腔医学, 2015, 24(6): 645-651.
[13]	苑克勇,马瑞,朱彩莲,李鸣宇,何智妍,周薇. 冷光美白对釉质表面细菌生物膜形成的影响[J]. 上海口腔医学, 2015, 24(3): 283-287.
[14]	王玉霞, 高丽, 江文欣, 马瑞, 唐子圣, 朱彩莲, 何智妍, 黄正蔚. 变异链球菌LuxS缺陷株甲基循环通路的恢复构建[J]. 上海口腔医学, 2014, 23(4): 385-390.
[15]	武侠, 孙静, 褚昊月, 亓庆国. 饥饿状态对白念菌生物膜及滞留菌形成的影响[J]. 上海口腔医学, 2014, 23(3): 285-289.