临床分离MRSA中氨基糖苷类抗生素磷酸化酶的克隆表达与活性测定

Cloning,over-expression and in vitro activity of aminoglycoside phosphotransferase from clinical MRSA isolates

目的:从临床分离的耐甲氧西林金黄色葡萄球菌(MRSA)中克隆氨基糖苷类抗生素磷酸化酶基因并异源表达,建立其磷酸化反应的体外测活方法。方法:考察临床分离MRSA对氨基糖苷类抗生素的敏感性,从中克隆磷酸化酶基因,并连入pET28a构建表达载体和菌株,将表达的重组蛋白以亲和色谱分离纯化;使用ESI-MS以及抗菌活性实验测定其磷酸化酶活性。结果:药敏实验表明临床分离的6株MRSA均对氨基糖苷类抗生素不敏感,重组表达克隆到的磷酸化酶,表达产物纯化后的纯度大于90%,体外催化试验表明,重组磷酸化酶2h内可以催化反应体系内的卡那霉素B完全磷酸化。结论:异源表达的氨基糖苷类抗生素磷酸化酶具有很好的活性,体外测活方法可靠快捷,为建立氨基糖苷类抗生素磷酸化酶抑制剂筛选模型打下基础。

Aim：To clone and over-express the gene encoding aminoglycoside（AG）phosphotransferase（APH）from clinical MRSA isolates in E.coli and to develop an assay method for the recombinant APH.Methods：The susceptibility of clinical MRSA isolates to AGs was tested by disk diffusion.A nucleic acid sequence encoding APH was amplified from the genomic DNA of an isolate and ligated to expression vector pET-28a,and then transformed into E.coli BL21（DE3）.After purification of the recombinant protein by affinity chromatography,the phosphorylation activity of the enzyme was determined by ESI-MS and disk diffusion.Results：All 6 clinical MRSA isolates were unsusceptible to AGs.After cloning and expression,the recombinant APH was purified to 90%.The in vitro activity assay indicated that the recombinant protein could inactivate kanamycin B in the assay mixture within 2 h.Conclusion：The recombinant APH showed excellent enzymatic activity.The assay method was simple and convenient,which may provide the basis of developing a screening model for APH inhibitors.