大肠杆菌外膜蛋白ompW基因敲除菌的构建及其对两种抗生素的敏感性

摘要

探讨大肠杆菌ompW基因敲除后,硫酸新霉素和氨苄青霉素对敲除菌最低抑菌浓度(MIC)和生存率的影响,进而分析OmpW的功能.[方法]运用Red重组技术将大肠杆菌K12染色体上基因ompW敲除,构建缺陷株△ompW.然后分别测定硫酸新霉素和氨苄青霉素对正常菌和△ompW菌的最小抑菌浓度(MIC)及次抑菌浓度(1/2 MIC)下K12和△ompW菌的生存率.[结果]经PCR鉴定和通过提取膜蛋白进行western blot 分析表明,成功获得ompW敲除菌.抗生素分析表明,K12菌对硫酸新霉素的MIC为8.0 μg/mL,生存率为98.0％；△ompW菌对新霉素的MIC为1.7 μg/mL,而其生存率仅为39.0％.而k12对氨苄新霉素的MIC为16.0 μg/mL,△ompW为3.3 μg/mL;1/2 MIC下K12生存率为70.4％,而△ompW为30.3％.[结论]ompW基因缺陷株对两抗生素的敏感性大大增强,表明ompW在细菌抗性方面起着关键作用.%[Objective] To investigate the contribution of an outer membrane protein OmpW to tolerance neomycinsulphate and ampicillin of Escherichia coli K12.[Methods] The ompW knock-out mutant (△ompW) of E.coli K12 was generated using λ-Red recombination system.Then the minimal inhibitory concentration (MIC) and the survival rates under 1/2 MIC of neomycinsulphate or ampicillin of △ompW and E.coli K12 were determined respectively.[Results] The △ompW was successfully obtained through confirmation of PCR analysis at the gene level and Western blot analysis at the protein level.The MIC of neomycinsulphate of △ompW is 1.7 μg/mL.The value is much lower than that of E.coli K12, which is 8.0 μg/mL.Difference of survival rates under 1/2 MIC of neomycinsulphate of △ompW and E.coli K12 was also observed, and their survival rates are 39% and 98% , respectively.The MIC of ampicillin of △ompW is 3.3 μg/mL.The value is also lower than that of E.coli K12 ( 16.0 μg/mL) .The survival rates under 1/2 MIC ampicillin of △ompW and E.coli K12 are 30.3% and 70.38% , respectively.[ Conclusion ] The △ompW is much more sensitive to neomycinsulphate and ampicillin than its parent strain.The result indicated that OmpW played crucial role in bacteria resistance of drug.