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

摘要:

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;1/2MIC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。

Abstract:

Abstract:［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 8.0μg/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 (16.0μg/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．