志贺菌外排泵基因emrE克隆表达及进化分析.doc

志贺菌外排泵基因emrE克隆表达及进化分析 　 作者：吴健 张少平 穆瑞瑞 丁毅 郗园林 段广才 【摘要】 目的 研究志贺菌外排泵基因emrE的耐药功能及其进化。方法 以志贺菌临床多重耐药株H24基因组为模板，扩增出含emrE基因的1126bp DNA片段，将所得片段与pMD18-T载体连接，转化大肠埃希菌DH5α，对阳性克隆酶切鉴定和测序；用琼脂平皿二倍稀释法对重组菌株进行药敏测定并测定泵抑制剂CCCP对MIC值的影响；通过RT-PCR从mRNA水平检测emrE的表达水平；通过Genbank数据库对基因序列进行同源性比对，建立进化树并分析同源基因之间的关系。结果 含emrE基因质粒可以提高大肠埃希菌对四环素耐药性1倍，对红霉素的耐药性1倍；对emrE基因的同源分析显示H24菌株和大肠埃希菌、其它志贺菌可归为相近一族，同源性在92%以上，与同属于肠杆菌科的欧文杆菌和沙门菌的同源性分别为70%和60%；与亲缘关系较远革兰阳性菌除虫链霉菌和结核分枝杆菌的同源性仅为47%和48%。结论 志贺菌耐药株H24的emrE基因与对四环素及红霉素的耐药性相关，依据现有数据进行的emrE基因同源分析未发现有明显的基因水平转移现象。 【关键词】 志贺菌； emrE基因； 外排泵； 耐药； 基因水平转移 ABSTRACT Objective To study the function and phylogenetics of the active efflux pump gene emrE in clinical isolates of Shigella. Methods The active efflux pump gene emrE was amplified by PCR and ligated with the pMD18-T plasmid to construct the recombinant pMD-emrE, and the pMD-emrE was transformed into E.coli DH5α. The positive bacterial clones were identified by restriction enzyme digestion, and the gene of isolates was sequenced and analyzed. Drug susceptibility of the recombinant strain was tested by the two fold agar dilution method; phylogenic tree was constructed with the data of the gene sequences from Genbank. Results Plasmid pMD-emrE can elevate the resistance level of E.coli to tetracyclin by one fold and erythromycin by one fold. Analysis of emrE phylogenic tree indicated that strain H24, all strains of E.coli and all other strains of Shigella could be classified into one group with 92% gene sequence identity. Strains of Erwinia and Salmonella, which also beloned to Enterobacteriaceae, had 70% and 60% sequence identity with H24. Streptomyces avermitilis and Mycobacterium tuberculosis were only 47% and 48% sequence identity with H24. Conclusions The gene emrE was related to the resistance of tetracycline and erythromycin in the drug resistance strain H24. Phylogenic tree analysis indicates it was scarce for emrE to develop horizontal gene transferring among different bacterial genomes. KEY WORDS Shigella;emrE;Act