肺炎链球菌绿色荧光蛋白报告质粒的构建及评价  

Construction and evaluation of a high effective GFP reporter plasmid for Streptococcus pneumoniae

作　　者：胥文春[1] 单幼兰[2] 许颂宵[1] 王虹[1] 陈淑惠[1] 尹一兵[1] 

机构地区：[1]重庆医科大学检验系临床检验诊断学省部共建教育部重点实验室、重庆市重点实验室,重庆400016 [2]重庆医科大学附属第二医院传染病实验室,重庆400012

出　　处：《重庆医科大学学报》2008年第12期1409-1413,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(30471838,30400376)

摘　　要：目的:构建一个可高效报告基因表达的绿色荧光蛋白(Green fluorescence protein,GFP)报告质粒,为采用差异荧光诱导(Differential fluorescence induction,DFI)技术筛选肺炎链球菌(Streptoccus pneumoniae,S.pn)体内诱导的毒力基因奠定基础。方法:酶切质粒pGreenTIR的SD-ENH-GFP片段,将其插入含氯霉素抗性基因的自杀质粒pEVP3中,构建以绿色荧光蛋白为报告基因的自杀质粒pEVP3-SDGFP。再将肺炎链球菌的溶血素基因(Pneumolysin,ply)上游约500bp片段插入到该质粒报告基因上游的多克隆位点中,得到质粒pEVP3-SDGFP-Ply,将其转化入肺炎链球菌TIGR4中,比较新建质粒pEVP3-SDGFP与同样处理质粒pEGFP-1(报告基因gfp前无SD及ENH序列)报告上游基因表达的情况。结果:通过体内、外实验证实新建质粒pEVP3-SDGFP不但可以报告肺炎链球菌溶血素基因的表达,并且其报告上游基因表达的能力明显强于质粒pEGFP-1。结论:所构建的质粒pEVP3-SDGFP可用于DFI筛选肺炎链球菌体内诱导基因所需启动子诱捕文库的构建。Objective： To construct a high effective GFP reporter plasmid for screening Streptoccus pneumoniae virulent genes by differential fluorescence induction. Methods： The SD-ENH-GFP region of plasmid pGreenTIR was cloned into a suicide plasmid pEVP3 which contains a cat gene encoding resistance protein to chloramphenicol,and a report plasmid pEVP3-SDGFP was constructed.To evaluate the function of this plasmid, a 500bp fragment of the pneumolysin gene （ply） of TIGR4 was coloned in the upstream of gfp and then was transformed into Streptococcus pneumoniae TIGR4. Results： The plasmid pEVP3-SDGFP could report the expression of ply both in vivo and in vitro,and was more effective than plasmid pEGFP-I without SD and ENH sequence before gfp gene. Conclusion： Plasmid pEVP3-SDGFP can be used to construct the promoter-trap library which is needed in DFI.

关 键 词：肺炎链球菌 绿色荧光蛋白 报告质粒 

分 类 号：R392.1[医药卫生—免疫学]