临床分离MRSA中氨基糖苷类双功能修饰酶AAC(6)′-APH(2″)的克隆表达与活性测定

Cloning,overexpression and assay of the aminoglycoside bifunctional modifying enzyme AAC(6′)-APH(2″) from MRSA clinical isolates

双功能酶AAC(6′)-APH(2″)是一种重要的氨基糖苷类抗生素钝化酶,从临床分离出的6株耐甲氧西林金黄色葡萄球菌(MRSA)的总DNA中克隆得到AAC(6′)-APH(2″)基因,将该基因插入质粒pET-28a中构建表达载体,然后将该重组质粒转入大肠杆菌BL21进行异源表达,在IPTG的诱导下产生大量可溶性目标重组蛋白,该重组蛋白经亲和层析分离纯化,SDS-PAGE鉴定纯度大于90%;建立了此双功能酶的体外测活方法,为建立氨基糖苷类抗生素双功能酶抑制剂筛选模型奠定基础.

Bifunction enzyme AAC（6′）-APH（2″） is an important passivation enzyme of aminoglycosides（AGs）.The chromosome gene encoding AG bifunctional enzyme from the 6 MRSA clinical isolates was cloned and introduced into expression plasmid pET-28a,and then was transformed into E.coli BL21（DE3）.The strains produced large amounts of soluble recombinant protein under IPTG induction.The protein was purified by affinity chromatography.After purification,the purity of recombinant AAC（6′）-APH（2″） reached 90%.A method to assay the recombinant enzyme activity was developed in vitro,which laid a good basis for developing a screening model for obtaining the AAC（6′）-APH（2″） inhibitors.