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

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.

Construction of ompW knock-out mutants of Escherichia coli to increase sensitivity to neomycinsulphate and ampicillin

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(ΔompW) 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 ΔompW and E.coli K12 were determined respectively.Results] The ΔompW 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 ΔompW 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 ΔompW and E.coli K12 was also observed,and their survival rates are 39% and 98%,respectively.The MIC of ampicillin of ΔompW 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 ΔompW and E.coli K12 are 30.3% and 70.38%,respectively.Conclusion] The ΔompW 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.