金黄色葡萄球菌N-乙酰甘露糖胺-6-磷酸2-异构酶基因的克隆与表达

The cloning and expression of N-acetylmannosamine-6-phosphate 2-epimerase gene from Staphylococcus aureus

目的对金黄色葡萄球菌N-乙酰甘露糖胺-6-磷酸2-异构酶基因(APE)进行克隆、鉴定与表达,为新型抗菌药物的设计提供参考依据。方法采用聚合酶链反应(PCR)扩增APE基因,将扩增产物酶切后与原核表达载体pET-32a(+)连接,构建表达APE的重组质粒,将重组质粒先转化大肠杆菌TG1内并提取质粒,经PCR、双酶切鉴定后再转化表达宿主大肠杆菌BL21(DE3),对转化菌株进行诱导后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。结果构建了表达载体pET-32a(+)-APE,在37℃经异丙基-β-D硫代半乳糖苷诱导时获得表达,表达的蛋白质相对分子质量为45.2×10~3。结论 APE基因成功克隆到表达质粒内并获得了表达。

Objective To clone,identify,and express the N-acetylmannosamine-6-phosphate 2-epimerase（APE）gene from Staphylococcus aureus in order to provide reference data for the design of new antibacterial drugs.Methods APE gene was amplified with polymerase chain reaction（PCR）from Staphylococcus aureus,inserted into pET-32 a（＋）,transformed into Escherichia coli TG1,and plasmid DNA was extracted,and digested with restriction endonucleases.The right insertion was retransformed into E.coli BL21（DE3）.The cultures were loaded onto SDS-PAGE.Results The expression vector pET-32 a（＋）-APE was constructed and expressed in the presence of isopropyl-beta-D galactosyl thioglycoside at 37℃,The relative molecular mass of the expressed protein was 45.2×10~3.Conclusion The APE gene was successfully cloned into the expression plasmid and obtained the expression.