摘要
[目的]对金黄色葡萄球菌一种新的青霉素结合蛋白(PBPX)进行原核表达,为新型抗菌药物设计提供参考数据。[方法]将ATCC25923基因组数据通过Blast进行比对,从中发现了一个含有PBP保守结构域的基因序列,命名为PBPX。PCR扩增PBPX基因,将扩增产物酶切后与原核表达载体p ET-32a(+)连接,构建表达PBPX的重组质粒,将该重组质粒转化大肠杆菌TG1内,增菌培养后提取质粒,经PCR、双酶切鉴定后,再转化大肠杆菌BL21(DE3),对转化菌株诱导后进行10%SDS-PAGE分析。[结果]构建了表达载体p ET-32a(+)-PBPX,在37℃经异丙基-β-D硫代半乳糖苷(IPTG)诱导后获得表达,重组青霉素结合蛋白分子量为52. 2 k Da。[结论]PBPX基因成功克隆到表达载体内并获得了表达。
[ Objective ] To clone, identify, and express the penicillin -binding protein （PBPX）gene from Staphylococcus aureus in order to provide reference data for the design of new antibacterial drugs. [ Methods ] PBPX gene was amplified with polymerase chain reaction（PCR） from Staphylococcus aureus, inserted into pET -32a （ ＋ ）. Then transformed into E. coli TG1, plasmid was extracted after bacterial culture, digested with restriction endonucleases. The right insertion was retransformed into E. coli BL21 （DE3）. The cultures were loaded onto 10% SDS -PAGE. [ Results] Both cloning and expression recombinants of PBPX were obtained. The rPBPX was expressed an apparent MW of 52.2 kDa when induced by IPTG at 37 ℃. [ Conclusion] The PBPX gene was successfully cloned into the expression plasmid and obtained the expression.
作者
李文通
盛佩群
羊晓敏
陈逸璐
何文迪
夏佳音
陈新江
Wentong Li;Peiqun Sheng;Xiaomin Yang;Yilu Chen;Wendi He;Jiayin Xia;Xinjiang Chen(Medical Technology Institute,Ningbo Health Vocational and Technical College,Ningbo 315100,China;School of Pharmaceutical Engineering,Zhejiang Pharmaceutical College,Ningbo 315100,China)
出处
《生物技术》
CAS
2018年第5期425-427,454,共4页
Biotechnology
基金
宁波市教育科学规划重点课题(No.2018YZD035)
浙江医药高等专科学校校级科研项目(No.ZPCSR2016008)
关键词
金黄色葡萄球菌
青霉素结合蛋白
药物靶点
克隆
表达
Staphylococcus aureus
penicillin - binding protein
drug target
cloning
expression
