利用靶定基质蛋白基因m的小干扰RNA抑制2009甲型H1N1流感病毒的复制

目的:设计、构建并筛选针对流感病毒基质蛋白基因m的小干扰RNA(siRNA),检测其对2009甲型H1N1流感病毒复制的抑制效果。方法:设计3条针对流感病毒m基因的siRNA,并克隆到短发夹型(shRNA)siRNA表达载体pSilencer2.1-U6-hygro上;经测序证明构建成功后,将表达3种siRNA的质粒和阴性对照质粒psiRNA-control分别转染MDCK细胞,用潮霉素筛选稳定表达细胞株,用H1N1流感病毒感染细胞,通过Real-time PCR和Western印迹检测干扰效果。结果:构建了3个针对流感病毒m基因的siRNA表达质粒,3种siRNA均使m基因的mRNA水平降低,其中M1-306的抑制效率达60%;3种siRNA均使流感病毒NA蛋白的表达量降低,M2-25、M1-105的抑制效率明显,M1-306略低。结论:针对m基因的siRNA可以有效抑制流感病毒在MDCK细胞中的复制。

Inhibition of 2009 Pandemic Influenza Virus H1N1 Replication by the Small Interfering RNA Targeting Matrix Gene

Objective： To design and construct the small interfering RNA（siRNA） targeting the matrix gene m of 2009 pandemic influenza virus H1N1 and evaluate their abilities to inhibit influenza virus replication.Methods： Three m-siRNAs were designed and inserted into pSilencer2.1-U6-hygro expression plasmid.After confirmation of the recombination by DNA sequencing,MDCK cells were transfected with the siRNA expression plasmid,selected for stably expressing the siRNA with hygromycin and infected with 2009 pandemic influenza virus H1N1.The suppression effects were identified by Real-time PCR and Western bolt.Results： The three m-siRNAs were obtained.All three siRNAs have significant suppression effects on the level of m-mRNA.The inhibition rate of M1-306 is about 60%.Western blot showed a reduction of NA protein expression,M2-25 and M1-105 have more significant suppression effects than M1-306.Conclusion： The constructed m-siRNAs could effectively inhibit the replication of influenza virus.