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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 vector targeting Rab9 GTPase gene and observe the inhibitory effect of shRNA on Rab9 GTPase expression and in vitro replication of measles virus Edmonston strain. Methods shRNA expression vector targeting Rab9 GTPase gene was constructed according to shRNA design and Rab9 GTPase gene sequence. The expression vector was transfected into Vero-E6 cells by restriction endonuclease digestion and sequence analysis, and then infected with Edmonston strain of measles virus. The expression of Rab9 GTPase mRNA and protein in transfected cells was detected by RT-PCR and Western blot. The virus titer was detected by standard plaque assay to observe the inhibitory effect of shRNA on the replication of Edmonston strain. Results The shRNA targeting Rab9 GTPase specifically inhibited the expression of Rab9 GTPase mRNA and protein in Vero-E6 cells with the maximum inhibitory rates of (86.3 ± 0.7)% and (87.6 ± 0.7)%, respectively. The plaque assay detected Rab9 GTPase After gene expression was silenced, the replication rate of Edmonston strain in vitro was more than 90%, and the inhibitory effect could last for at least 32 days. Conclusion shRNA targeting Rab9 GTPase can specifically inhibit the expression of Rab9 GTPase and the replication of Edmonston strain of measles virus in vitro, which is expected to become a nucleic acid drug for anti-measles virus infection.