肺炎克雷伯菌药物外排泵mdtl的克隆及分析

Cloning and Mechanism Analyzing of Drug Efflux Gene mdtl of Klebsiella pneumonia

[目的]探讨肺炎克雷伯菌ATCC 49790(KP)耐药的分子机制。[方法]以KP基因组DNA为模板来扩增mdtl基因,将mdtl基因扩增产物进行DNA测序及同源性分析;将扩增产物与载体连接后转化到大肠埃希菌KAM32感受态细胞中,对转化子进行药敏分析。[结果]KP的mdtl基因扩增产物与已报道的mdtl基因核苷酸序列同源性达到99%,这个转化子对红霉素、氯霉素和卡那霉素具有很高的耐药性,这种高耐药性能被外排泵抑制剂羰基氰氯苯腙和维拉帕米逆转。[结论]克隆到了KP一多药外排基因。

[Objective] The aim was to investigate the molecular mechanism of drug-resistance in Klebsiella pneumoniae ATCC 49790（KP）.[Method] PCR amplification of the drug resistance gene mdtl was carried out using chromosomal DNA of KP as template.PCR product of gene mdtl was sequenced.The nucleotide sequences and deduced amino acid sequences were analyzed by BLAST of NCBI.MICs of various drugs were determined by serial two-fold dilution method.[Result] PCR product of KP shared 99% identities with the mdtl in other bacterium which had been reported.The transformation had high resistance to erythromycin,chloramphenicol and kanamycin.The drug resistance of the transformant could be reversed by carbonyl cyanide m-chlorophenylhydrazone（CCCP） and verapamil.[Conclusion] The cloned gene was a multiple drug efflux gene.