摘要
目的 通过构建变形链球菌luxS基因高表达株,研究该菌的密度感应luxS基因对生物膜形成的影响.方法 PCR扩增变形链球菌luxS基因片段与双酶切的大肠杆菌-链球菌穿梭表达载体pIB169连接,构建pIB-luxS质粒;将其电击转化入变形链球菌,筛选出高表达株,经电泳、蛋白质印迹法鉴定构建成功.观察野生株和高表达株的生长曲线（A600值）;通过甲基噻唑基四唑法比较两种菌株在不同时间点及培养基中含不同质量分数（0、0.125％、0.25％、0.5％）蔗糖时的生物膜形成量（A590值）,激光扫描共聚焦显微镜观察生物膜结构,实时PCR检测相关基因的表达（以野生株为对照组）,实验均重复3次.结果 luxS片段插入正确,目的蛋白表达,高表达株成功构建,且体外稳定传代.变形链球菌野生株及高表达株在浮游模式的生长曲线无差异,但高表达株细菌在14和24 h时生物膜A590值分别为0.400±0.009和0.609±0.041,均显著高于相同时间点野生株（A590值分别为0.352±0.028和0.533±0.014）（P＜0.05）.当加入0.125％蔗糖时,变形链球菌高表达株A590值为1.041±0.038,显著高于野生株（0.831±0.020）（P＜0.05）;随着蔗糖质量分数增加,两菌株的生物膜量均增加,但两菌株间差异无统计学意义.高表达株在结构上集聚程度增加,形成团块,且其相关基因gtfB、ftf、gbpB、relA、brpA、smu630、comDE及vicR的相对表达量均显著高于对照组（P＜0.05）.结论 变形链球菌密度感应luxS基因促进细菌生物膜的形成.
Objective To evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans（Sm）.Methods In order to construct pIB-luxS plasmid,the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests.The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot.The growth rate of both Sm wild type strain and its luxS overexpression strain were observed.Methyl thiazolyl tetrazolium（MTT） assay method was used to compare the biofilm formation quantificationally by both strains at different time points and containing different sucrose.The structures of the biofilms were observed by using confocal laser scanning microscopy,and biofilm-related gene expressions were investigated by real-time PCR.All experiments were performed in triplicate.Results The luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting.The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference,but biofilm formed by Sm overexpression strain was 0.400 ±0.009 and 0.609±0.041 at 14 and 24 h,higher than the wild type strain biofilm at the same time point（0.352 ±0.028 and 0.533 ±0.014,respectively,P〈 0.05）.After adding 0.125% sucrose,biofilm formed by Sm overexpression strain raised to 1.041 ±0.038,higher than that by the wild type strain（0.831 ±0.020,P〈0.05）.The biofilm formed by both strains were also increased with the sucrose concentration increase,but there was no difference between them.The overexpression strain aggregated into distinct clusters on structure,genes expression including gtfB,ftf,gbpB,relA,brpA,smu630,comDE,vicR were increased（6.10±0.12,3.34±0.07,8.75±0.13,2.96±0.04,5.20± 0.19,2.20±0.06,2.32±0.07 and 10.67±0.57 fold） compared to the wild-type strain（P〈0.05�
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2015年第9期554-560,共7页
Chinese Journal of Stomatology
基金
国家自然科学基金（81300866、81371143、81370024）
关键词
链球菌
变异
生物膜
基因表达
Streptococcus mutans
Biofilm
Gene expression
