小发卡RNA抗呼吸道合胞病毒效应的研究

目的 为从基因水平抑制呼吸道合胞病毒（respiratory syncytial virus，RSV）的复制，针对RSV的M2-2基因构建小发卡结构状RNA（short hairpin RNA，shRNA）重组质粒，在细胞水平观察shRNA对RSV复制的影响。方法 将成功构建的针对RSV M2-2基因的重组质粒pshRNA8260转染HEp-2细胞，利用光镜观察pshRNA8260对RSV致HEp-2细胞病变效应（cytopathogenic effect，CPE）的影响并计算CPE抑制率，空斑形成实验检测RSV滴度变化。结果 成功构建了针对人RSV M2-2基因mRNA的pshRNA8260重组质粒，研究发现pshRNA8260能明显改善RSV所致的病变效应，降低RSV在细胞内复制的病毒滴度。结论 针对RSV M2-2基因的pshRNA82（g）重组质粒具有明显的特异性抗RSV效应的作用。

The specificically effect of short hairpin RNA on the replication of respiratory syncytial virus

Objective In order to inhibit the replication of respiratory syncytial virus (RSV ) on gene level, the recombinant plasmid targeting M2-2 gene of RSV was constructed and the effect of RSV replication inhibition by specific RNAi was observed in this study. Methods The M2-2 gene which plays an important role on RSV replication and the target sequences which is easy to access as the targeting sites of RNAi were chosen, and the recombinant plasmid pshRNA8260 targeting the mRNA of RSV M2-2 gene coding sequence was constructed. The pshRNA8260 was transfected into HEp-2 cells. Microscopy was used to observe the effects of the pshRNA8260 on cytopathogenic effect(CPE) of HEp-2 cell induced by RSV infection and the inhibition ratio of CPE induced by pshRNA8260 was calculated. Viral plaque forming assay was used to detect the viral titer. Results The recombinant plasmid pshRNA8260 which targets the mRNA of RSV A/2-2 gene successfully constructed. The study showed that the pshRNA8260 significantly ameliorated CPE of RSV-infected HEp-2 cells, and reduced the viral titer of RSV replication in infected cells. Conclusions The recombinant pshRNA8260 plasmid targeting mRNA of RSV M2-2 gene has a significant and specific anti-RSV effect.