siRNA靶向抑制铜绿假单胞菌oprM基因表达

Suppressed expression of oprM by target siRNA in Pseudomonas Aeruginosa

目的探讨siRNA能否抑制铜绿假单胞菌(Pseudomonas aeruginosa,PA)主动外排系统oprM基因的表达。方法针对oprM基因序列设计合成3对siRNA片段,分别转化耐药PA,采用抗菌素药物敏感试验筛选出能抑制PA耐药的siRNA片段及其最佳转化剂量,再构建siRNA质粒表达载体,转化PA标准菌株,应用western blot技术检测OprM蛋白表达量的变化。结果筛选出一对能抑制PA耐药的siRNA片段,确定其最佳转化剂量为25μL(20μm/L)/100μL PA菌液。成功构建了靶向oprM基因的siRNA质粒表达载体。Western Blot检测结果显示,转化siRNA质粒载体的PA其OprM蛋白的表达量明显降低。结论靶向oprM基因的siRNA能够抑制PA oprM基因的表达。

Objective To investigate whether the expression of active efflux system oprM gene can be inhibited by target siRNA in pseudomonas aeruginosa （PA）. Methods Three pairs of siRNAs were designed and synthesized according to the reference sequence of oprM gene, and then transformed into the competent drug-resistance PAs respectively. The most effective siRNA and optimal transformation dosage were screened out by antimicrobial susceptibility test. The plasmid expression vector containing the effective siRNA fragment was constructed and then transformed into standard PA strain. The amount of protein expression levels of oprM was tested by western blot. Results An effective siRNA was screened out and the optimal transformation dosage was ascertained to be 25 μL （20 μm/L）/100 μL PA. The oprM gene targeted siRNA- expression plasmid vector was successfully constructed. The results of western blot showed that the expression of OprM protein in PA transformed with siRNA-expression plasmid vector decreased significantly. Conclusion The expression of the oprM gene in PA can be inhibited by oprM targeted siRNA.