肺炎克雷伯菌耐庆大霉素钝化酶基因的克隆及鉴定

Molecular cloning and characterization of gentamicin-N-acetyltransferase (3)-V gene from a K. pneumoniae isolate in Huashan hospital

近年的细菌耐药性监测结果发现，肺炎克雷伯菌对庆大霉素等氨基糖苷类抗生素的耐药率已达４０％左右。耐药的主要机制为细菌产生Ｎ－乙酰转移酶ＡＡＣ（３）－Ｖ，使其抗菌活性不能发挥。不同菌株产生的钝化酶及其编码基因有一定的差异。作者从华山医院分离的肺炎克雷伯菌耐药株，用ＰＣＲ方法从该菌株的一分子量约８５×１０＾３的接合型质粒上扩增出全长基因，连接ｐＵＣ１８。

For the purpose of crystallography and kinetic study of N-acetyltranferase (3)-V , the encoding gene , aac (3)-Va was cloned from K.pneumonia and expressed in E. coli. The aac (3) -Va amplified from a conjugate plasmid of 85Md by PCR was ligated in pUC 18. From the transformants ,we isolated the clone encoding N-acetyltransferase (3)-V. It was proved by restriction fragments analysis and sequencing, more than 95％ sequence of this gene was identical to that of S. marcescens, Most of the amino acids substitution located in C terminal. This gene ligated with pLY4 was successfully expressed in E. coli JF1125 and DH5a.