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

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

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ΔompW。然后分别测定硫酸新霉素和氨苄青霉素对正常菌和ΔompW菌的最小抑菌浓度(MIC)及次抑菌浓度(1/2 MIC)下K12和ΔompW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8.0μg/mL,生存率为98.0%;ΔompW菌对新霉素的MIC为1.7μg/mL,而其生存率仅为39.0%。而k12对氨苄新霉素的MIC为16.0μg/mL,ΔompW为3.3μg/mL;1/2 MIC下K12生存率为70.4%,而ΔompW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（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.