变异链球菌LuxS缺陷株甲基循环通路的恢复构建

上海口腔医学 ›› 2014, Vol. 23 ›› Issue (4): 385-390.

• 基础研究 • 下一篇

变异链球菌LuxS缺陷株甲基循环通路的恢复构建

王玉霞, 高丽, 江文欣, 马瑞, 唐子圣, 朱彩莲, 何智妍, 黄正蔚

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

收稿日期:2014-01-21 出版日期:2014-08-20 发布日期:2014-10-20
通讯作者: 黄正蔚,Fax:021-63135412,E-mail:huang_zhengwei@hotmail.com
作者简介:王玉霞(1987- ), 女, 学士, E-mail:wxy870111@163.com
基金资助:
国家自然科学基金(81070826,81371143,81300866); 上海市启明星计划(12QH1401400)

Complementation and function of methyl-metabolic pathway in Streptococcus mutans luxS null strain

WANG Yu-xia, GAO Li, JIANG Wen-xin, MA Rui, TANG Zi-sheng, ZHU Cai-lian, HE Zi-yan, HUANG Zheng-wei

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

Received:2014-01-21 Online:2014-08-20 Published:2014-10-20
Supported by:
Supported by National Natural Science Foundation of China (81070826, 81371143, 81300866) and partly supported by Shanghai Rising-Star Program (12QH1401400).

摘要/Abstract

摘要： 目的:以变异链球菌LuxS缺陷株为模型,构建其密度感应缺陷的甲基循环恢复株。方法:以LuxS为阳性表达对照,通过在变异链球菌LuxS缺陷株中异源表达SahH,实现其代谢通路的恢复构建。通过Western 印迹、反转录PCR和信号分子AI-2分泌检测,验证目的蛋白的表达。采用SAS8.0软件包对数据进行统计学分析。结果:Western 印迹检测2种蛋白在大肠杆菌中均得到正确表达,验证了克隆载体的正常表达能力;反转录PCR验证了变异链球菌LuxS缺陷株中luxS及sahH mRNA的存在;发光实验证实LuxS阳性表达对照株AI-2的分泌能力恢复。结论:成功构建变异链球菌LuxS缺陷的代谢恢复株,为探讨LuxS的调控机制提供了分子生物学工具及实验对照。

关键词: 变异链球菌, 生物膜, 密度感应, LuxS, SahH

Abstract: PURPOSE: To complement the activated methyl cycle (AMC) pathway at an AI-2 defect background in Streptococcus mutans (S. mutans) luxS null strain. METHODS: A sahH gene was amplified from Pseudomonas aeruginosa and introduced into the S. mutans luxS null strain to complement the methyl-metabolic disruption at an AI-2 defect background. Western blot, reverse-transcription PCR and AI-2 bioassay were performed to confirm the heterogenous expression of SahH in S. mutans luxS null strain. The data was statistically analyzed by SAS8.0 software package. RESULTS: LuxS and SahH were detected to express in Escherichia coli BL21 as well as their mRNA were confirmed to be successfully transcribed in S. mutans luxS null strain. AI-2 production was found in wide type S. mutans and its luxS-introduced luxS null strain but not found in the luxS null strain and its sahH and empty plasmid-introduced strains. CONCLUSIONS: A new S. mutans derivative with the AMC pathway complements while the AI-2 defect is constructed.

Key words: Streptococcus mutans, Biofilm, Quorum sensing, LuxS, SahH

中图分类号:

R780.2

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.

WANG Yu-xia, GAO Li, JIANG Wen-xin, MA Rui, TANG Zi-sheng, ZHU Cai-lian, HE Zi-yan, HUANG Zheng-wei. Complementation and function of methyl-metabolic pathway in Streptococcus mutans luxS null strain[J]. Shanghai Journal of Stomatology, 2014, 23(4): 385-390.

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变异链球菌LuxS缺陷株甲基循环通路的恢复构建

Complementation and function of methyl-metabolic pathway in Streptococcus mutans luxS null strain

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