靶向Rab9 GTPase基因shRNA体外抑制麻疹病毒Edmonston株复制的研究  

Study on inhibiting the replication of the Edmonston strain of the measles virus using short hairpin RNA targeting the Rab9 GTPase gene in vitro

作　　者：史俊岩[1] 王美莲[1] 刘兵[1] 罗恩杰[1] 

机构地区：[1]中国医科大学基础医学院病原生物学教研室,辽宁沈阳110001

出　　处：《中国病原生物学杂志》2011年第3期164-167,共4页Journal of Pathogen Biology

基　　金：辽宁省教育厅基金项目(No.2008758)

摘　　要：目的构建靶向Rab9 GTPase基因的短发夹RNA(shRNA)表达载体,观察shRNA对Rab9 GTPase表达和麻疹病毒Edmonston株体外复制的抑制效应。方法按照shRNA的设计要求和Rab9 GTPase基因序列构建靶向Rab9 GTPase基因shRNA表达载体,表达载体经酶切鉴定和序列分析后通过脂质体法转染Vero-E6细胞,然后感染麻疹病毒Edmonston株,通过RT-PCR和Western blot检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;通过标准蚀斑试验检测病毒滴度,观察shRNA对麻疹病毒Edmonston株复制的抑制效应。结果靶向Rab9 GTPase基因的shRNA可特异性抑制Vero-E6细胞内Rab9 GTPase mRNA和蛋白质的表达,最大抑制率分别为(86.3±0.7)%和(87.6±0.7)%;蚀斑试验检测Rab9 GTPase基因表达沉默后对麻疹病毒Edmonston株体外复制抑制率达90%以上,且抑制效应至少可持续32 d。结论靶向Rab9 GTPase基因的shRNA能特异性抑制Rab9 GTPase表达和麻疹病毒Edmonston株体外复制,有望成为抗麻疹病毒感染治疗的核酸药物。Objective To construct short hairpin RNA（shRNA） expression vectors targeting the Rab9 GTPase gene and to assess the inhibitory effect of shRNA on the expression of Rab9 GTPase and replication of the Edmonston strain of the measles virus in vitro.Methods A short hairpin RNA expression vector targeting the Rab9 GTPase gene was constructed in accordance with requirements for shRNA design and the Rab9 GTPase gene sequence.shRNA were transfected into Vero-E6 cells via liposomes,and then cells were infected with the Edmonston strain of the measles virus.The expression of Rab9 GTPase mRNA was assayed by RT-PCR,and the expression of Rab9 GTPase protein was observed using a Western blot assay.ShRNA-mediated inhibition of measles virus replication was studied by detecting the titer of the measles virus.Results Compared to the control group,shRNA targeting the Rab9 GTPase gene significantly inhibited the expression of Rab9 GTPase mRNA and protein in Vero-E6 cells,with a maximum inhibition rate of（86.3±0.7）% and（87.6±0.7）%,respectively.Measles virus replication was suppressed dramatically and consistently by shRNA targeting the Rab9 GTPase gene;most importantly,this suppression persisted after 32 d.Conclusion shRNA targeting the Rab9 GTPase gene specially inhibited the expression of Rab9 GTPase and measles virus replication in vitro;these shRNA can potentially be developed into therapeutic agents for the treatment of measles virus infection.

关 键 词：Rab9 GTPASE SHRNA 麻疹病毒Edmonston株 复制 

分 类 号：R373.11[医药卫生—病原生物学]