目的 研究膜转运蛋白NorA的转运机制以及抑制剂对其转运机制的影响.方法 将NorA重组表达在大肠埃希菌中,采用微孔板法测定表达菌株对于数种奎诺酮类抗生素的最小抑菌浓度;后通过对比NorA过量表达菌株与对照组在诺氟沙星和/或NorA抑制剂胡麻碱存在时的生长情况,对其转运功能以及抑制剂对转运功能的阻碍进行研究;最后将在大肠埃希菌中过量表达的NorA采用去污剂进行分离纯化.结果 NorA表达菌株对于诺氟沙星和环丙沙星的最小抑菌浓度明显降低,但对司氟沙星的敏感度不变.NorA抑制剂胡麻碱的存在可显著阻断NorA的转运功能.NorA在大肠埃希菌中被成功表达纯化,产量可达到0.5~1 mg/L培养物,且纯度可达90%.这为治疗由于NorA过量表达导致的耐甲氧西林金黄色葡萄球菌的感染治疗提供了借鉴意义,同时为今后对NorA的功能研究奠定了基础.结论 NorA的表达可导致细菌对奎诺酮类抗生素的耐药,但其抑制剂胡麻碱可阻断NorA的转运功能.%Objective To analyze the transport mechanisms of Nor A and the effects of its transport inhibitor piperine. MethodsrnFirstly,the minimal inhibitory concentrations of Norfloxacin, Ciprofloxacin and Sparfloxacin were determined in E. coli with over-expressed NorA by using micrplate method;then growth curve was adapted to test the effects of piperine on the susceptibility of NorA over-expressed E. coli to Norfloxacin;at last NorA was over-expressed in Escherichia. Coli and then was purified by immobilized metal affinity chromatography (IMAC),the purity was observed by SDS-PAGE. Results The expression of NorA could reduce the susceptibility of Escherichia. Coli to Norfloxac and Ciprofloxacin,but not Sparfloxacin in minimal inhibitory concentration test. This change in susceptibility could be converted by adding the inhibitor of NorA, piperine. Besides, NorA was successfully over-expressed in Escherichia. Coli. The yield was 0. 5～T mg/L culture and the purity was more than 90%. Conclusion NorA can actively transport the Norfloxac and Ciprofloxacin out of cell and its function can by inhibited by the presence of Piperine.
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