肺炎克雷伯菌耐氨基糖苷类抗生素基因aac（3）-I的改构与功能

研究氨基糖苷乙酰转移酶基因aac(3)-I碱基变异与功能的关系。构建表达载体pET28a-aac(3)-I,转化感受态细胞E.coli BL21(DE3),筛选到阳性克隆送测序。然后在体外对aac(3)-I基因进行易错PCR改造,筛选到一株阳性克隆测序后发现7处碱基变异,其中C272T、T373C引发了氨基酸的变异,分别对应:Ser91Phe、Tyr125His。以琼脂稀释法检测不同克隆的最低抑菌浓度(minimum inhibitory concentration,MIC)。实验结果表明变异株MIC较突变前下降64倍(庆大霉素)、4倍(阿米卡星)、8倍(依替米星)。为进一步了解和阐明耐药酶AAC(3)-I与底物的作用机制打下基础。

Reconstruction and Function of aac(3)-I,an Aminoglycoside Resistant Gene from Klebiella pneumoniae

To study on the mutation of aminoglycoside acetyltransferase gene aac(3)-I and its function,expression vector pET28a-aac(3)-I was constructed,then transformed into competent E. coli BL21(DE3). One positive clone was sequenced. The aac(3)-I was reconstructed in vitro by error-prone PCR. A clone by antibiotics screening was sequenced. The result demonstrated that the gene had 7 bases mutate,on the site of C272T,T373C the amino acids have varied to Ser91Phe,Tyr125His. Agar dilution tests were performed to detect the minimum inhibitory concentration (MIC) of different clone strains. The MIC of the mutational clone was lower 64 times (gentamicin),4 times (amikacin),8 times (etimicin) than the wild strain. This study provided the fundaments to further comprehend and elucidate the mechanism of interaction between AAC(3)-I and substrate.