金黄色葡萄球菌外排蛋白qaca的优化表达研究刘彦伶1郭丹1.docVIP

金黄色葡萄球菌外排蛋白 QacA的优化表达研究 刘彦伶1 郭丹1# 夏晓影1 贾 蓓* （重庆医科大学附属第一医院感染科、重庆市传染病寄生虫病学重点实验室，重庆400016） 摘要 目的：提高金黄色葡萄球菌外排基因qacA在大肠杆菌中的表达水平，为深入研究其结构功能、设计有效外排抑制剂奠定基础。方法：以重组质粒pBluescript II SK(+)/qacA为模板，通过PCR方法扩增出带有AatII和NheI酶切位点的qacA基因，经相应酶切、纯化后将目的基因分别与同样进行酶切的质粒pET-coco1在T4连接酶的作用下连接；同时根据大肠杆菌偏爱密码子，由公司对qacA基因进行密码子优化，将合成的基因插入到pET-coco1载体中。重组表达质粒pET-coco1/qacA及密码子优化的pET-coco1/qacA转化大肠杆菌BL21(DE3)，经IPTG诱导，SDS分析表达情况，Western Blotting试验鉴定表达蛋白。结果： (1)酶切鉴定证实qacA基因及密码子优化的qacA基因已插入原核表达载体pET-coco1，重组表达质粒pET-coco1/qacA及密码子优化的pET-coco1/qacA已构建成功； (2)重组质粒pET-coco1/qacA转化大肠杆菌BL21(DE3)后经IPTG诱导成功地表达了QacA蛋白，其分子量约为55千道尔顿；优化后重组载体pET-coco1/qacA在大肠杆菌中得到高效表达；(3)Western Blotting试验显示QacA表达蛋白与合成的兔抗呈特异性免疫反应。结论：优化后的pET-coco1/qacA在大肠杆菌中得到高效表达，为进一步研究qacA生物学功能奠定了物质基础。 关键词 金黄色葡萄球菌；外排蛋白；QacA；表达 中图分类号：R378 文献标志码：A Study on optimization Expression of Staphylococcal efflux protein QacA LIU yan-ling1 GUO dan1# XIA xiao-ying1,JIA bei* (Key Laboratory of Infectious and Parasitic Diseases in Chongqing, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016，China) AbstractObjective To enhance the expressiom level of the active effux gene qacA in Staphylococcus aureus in Escherichia coli (E.coli),in aim of providing basis for the further studies on the construction and function of qacA gene and the study on designing effective inhibitor of efflux protein. Methods Using recombinant plasmid pBluescript II SK(+)/qacA as template, qacA gene with AatII and NheI sites was amplified through PCR method. aAfter objective genes and prokaryotic expression vector pET-coco1 digested with AatII and NheI simultanously, the purified objective genes were inserted into the compatible sites of prokaryotic expression vector pET-coco1, by using T4 DNA ligase; Reflecting E.coli codon usage, qacA gene was optimized by company simultanously,and codon-optimized qacA gene was cloned into pET-coco1 vector.The recombinant plasmids of pET-coco1/qacA and codon-optimized pET-coco1/qacA was transformed in