摘要
目的:通过将大肠杆菌BL21中MnSOD、FeSOD以及Cu/ZnSOD调控序列(包括启动子,-3518～+15 bp)与红色荧光蛋白(RFP)连接,构建成含SOD调控序列的红色荧光蛋白报告基因,用以研究不同SOD启动子对RFP的调控作用。方法:采用重组PCR技术,分别构建以MnSOD、FeSOD、Cu/ZnSOD启动子驱动的RFP报告基因载体,并将构建的融合基因通过T克隆转化入大肠杆菌,通过不同浓度Cd2+作用,分别诱导RFP表达。结果:正确构建了三种SOD-RFP融合基因,重组PCR结果与测序结果完全一致;该报告基因在室温静息状态下,红色荧光有微弱表达,经Cd2+诱导后,红色荧光亮度明显增强,其中MnSOD调控序列驱动的RFP表达最为明显。结论:该报告基因载体的成功构建,为研究SOD的基因表达调控机制和研制检测环境中污染物的微生物传感器提供了重要基础和工具。
Objective：To construct red fluorescent protein（RFP） reporter gene vectors driven by different SOD regulatory sequence in E.coli through fusing MnSOD,FeSOD,Cu/ZnSOD regulatory sequence and RFP gene in BL21 E.coli.Methods： Red fluorescent protein（RFP） reporter gene and MnSOD,FeSOD,Cu/ZnSOD regulatory sequence were fused using recombinant PCR technology.And then the SOD-RFP genes connected with pMD18-T plasmid were transfered into E.coli.The RFP expressed after stimulated by different concentrations Cd2＋ respectively.Results： Three SOD-RFP fusion genes were correctly constructed,which were proved by DNA sequencing;the reporter gene expressed a little of red flouresecent protein in the resting state at room temperature,but more red flouresecent intensity was obviously increased after Cd2＋ induction and SODA regulatory sequence was the most obviously RFP expression driven.Conclusion： The red fluorescent protein reporter gene vetors driven by different SOD regulatory sequence in E.coli have been constructed successfully with a sensitive response to Cd2＋ stimulation.This system provides an important basis and convenient tool for the further study of the regulatory mechanism of SOD gene expression and development of microbial sensor to detect the environmental pollutants.
出处
《温州医学院学报》
CAS
2012年第1期48-52,共5页
Journal of Wenzhou Medical College
