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目的对临床耐多药志贺菌株H24的1种小多重耐药性家族(SMR)外排泵emrE基因进行定向分子进化,研究基因突变增强emrE外排泵耐药性的潜力,调查志贺菌外排泵基因emrE基因垂直进化对临床耐药发展的影响。方法通过用体外易错PCR(EP-PCR)方法对emrE基因进行随机突变实验,用微量稀释生长曲线法进行菌株的耐药性筛选,用荧光实时定量RT-PCR测定基因表达情况,应用DNA分析软件DNAStar进行生物信息学分析。结果在31个突变子中筛选到了突变子ep2-emrE(DH5α),它的emrE基因有6个氨基酸突变,分别为Thr-28→Ala,Cys-39→Ser,Tyr-40→终止子,Ser-72→Arg,Leu-93→Phe,Ile-101→Phe。它不但增强四环素和红霉素的耐药能力,还产生新的氯霉素耐药能力。结论尽管emrE基因较易获得新增耐药性,但在志贺菌对四环素、红霉素等抗生素耐药性发展中,emrE基因的进化突变没有被筛选,表明有其他能力更强的耐药基因在突变进化中起作用。
Objective To investigate the molecular evolution of a small multidrug resistance (SMR) efflux pump emrE gene from clinical multidrug-resistant Shigella strain H24 to investigate the potential of gene mutation to enhance the resistance of emrE efflux pump. Effect of efflux pump gene emrE gene vertical evolution on clinical drug resistance development. Methods Random mutagenesis of emrE gene was performed by in vitro error-prone PCR (EP-PCR). The drug resistance of the strain was screened by micro-dilution growth curve. The gene expression was determined by real-time fluorescence quantitative RT-PCR. Software DNAStar for bioinformatics analysis. Results The mutant ep2-emrE (DH5α) was screened out from 31 mutants. The emrE gene has 6 amino acid mutations, which are Thr-28 → Ala, Cys-39 → Ser, Tyr-40 → terminator, Ser -72 → Arg, Leu-93 → Phe, Ile-101 → Phe. It not only enhances the resistance of tetracycline and erythromycin, but also produces new resistance to chloramphenicol. Conclusion Although the emrE gene is more likely to acquire new drug resistance, the evolutionary mutation of emrE gene was not screened in the development of antibiotic resistance to tetracycline and erythromycin in Shigella, indicating that there are other drug resistance Genes play a role in mutation evolution.