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Reconstruction and Function of aac(3)-I， an Aminoglycoside Resistant Gene from Klebiella pneumoniae
Chang-Seng Zha, Zhen Wang, Jian-Xin Lu, Yun-Bo Chen, Li-Lin Zou, Jing-Zhang Ji, Yin Peng, Zhen-Huan Ming
Zhejiang Provincial Key Laboratory of Medical Genetics, Institute of Cellular and Molecular Medicine, Wenzhou Medical College, Wenzhou 325035, China
Abstract: 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.
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