TolC表达量与大肠埃希菌K-12耐四环素的研究.docVIP

TolC表达量与大肠埃希菌K-12耐四环素的研究 作者：张丹凤 黄传钟 王三英 【摘要】 随着抗生素的开发与使用，细菌在对多种抗生素的适应过程中逐渐 发展 出对抗生素耐药的反应机制。TolC是药物排出转运体系的外膜成分，与AcrAB一起形成主要的药物排出泵，有关其表达量与耐药间的关系目前尚不清楚。试验将tolC克隆到pET-32a载体上进行诱导表达，镍柱纯化，免疫新西兰大白兔，获得14000的抗血清。Western blotting分析表明，TolC的表达量在耐四环素的大肠埃希菌K-12中比对照组提高50%。细菌TolC高表达试验发现，其MIC从100μg/ml提高到200μg/ml。结果说明TolC的表达量与四环素耐药性直接相关，提示细菌可以通过调节外膜蛋白的表达实现对抗生素的耐受。 【关键词】 TolC； 耐药性； 大肠埃希菌K-12； 四环素 ABSTRACT Association of TolC expression with resistance to tetracycline in Escherichia coli K-12 was studid. TolC was an outer membrane protEin, which was bound with AcrAB to form a critical multidrug efflux pump. However, association of TolC amount with drug resistance was ill-defined. In the study, tolC was cloned into pET-32a vector induced expression and its recombinant protEIns were purified by Ni-NTA. The purified recombinant proteins were used for preparing of anti-TolC. The prepared antiserum with titer 1∶4000 was utilized as the primary antibody in Western blotting. It showed that TolC amount was increased 50% in tetracycline-resistant E.coli K-12 compared to its original control strain. When TolC was overexpressed, the strain showed strong resistance to tetracycline with its MIC from 100μg/ml to 200μg/ml. These findings indicated that TolC amount was directly correlated to bacterial resistance to tetracycline, suggesting that bacteria mounted an adaptive feedback to antibiotics by regulating outer membrane protein expression. KEY WORDS TolC; Drug resistance; E.coli K-12; Tetracycline 随着人们对抗生素的过分依赖和滥用，耐药菌株迅猛发展，对人类健康构成严重的威胁。四环素通过与核糖体的30S亚单位结合以抑制细菌蛋白合成［1］而成为广谱抗生素，其抗菌谱包括许多革兰阳性和阴性菌、立克次体、支原体、衣原体、放线菌等。在20世纪50年代，细菌对四环素都非常敏感；1953年Flakow等［2］首次发现痢疾志贺菌S.dysenteriae对四环素产生耐药性。后来又发现志贺菌属Shigella对四环素、氯霉素和链霉素产生了多重耐药性［2～4］。目前认为，细菌耐四环素机制可以分成以下三类：①产生相应的水解及修饰酶以破坏药物活性；改变药物靶点的结构，使药物无法识别；阻止药物与靶点接触，其中包括调节膜通透性以及药物排出转运体系的作用，而这两者都与外膜蛋白的作用紧密相关［5］。药物排出转运体系是当前细菌耐药性研究关注的热点，如大肠埃希菌中的AcrAB-TolC系统［6］和铜绿假单胞菌中的MexAB-OprM系统［7］等。药物排出转运体系是对抗生素开发及应用的一个挑战，深入研究其机制将有助于我们发现新的抗菌机制、找到新的靶点。AcrAB-TolC排出系统是大肠埃希菌的主要多重药物外排系统，