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prM蛋白是登革病毒膜蛋白M的前体,膜蛋白M对病毒的组装与成熟有重要作用,针对prM基因设计的小干扰RNA(siRNA)可短期抑制登革病毒复制.为了达到长期抑制登革病毒的效果,本研究构建了插入prM siRNA序列的重组慢病毒,利用流式细胞术分选以及杀稻瘟霉素抗性,筛选出稳定表达prM siRNA的非洲绿猴肾细胞(Vero细胞)系.经逆转录PCR及测序验证siRNA序列表达正确.Vero细胞中prM siRNA的表达率约为97.6%.当受到登革病毒攻击时,表达prMsiRNA的Vero细胞能够明显抑制登革病毒prM基因的表达,并抑制登革病毒在Vero细胞中的复制.建立的Vero细胞系可用于RNA干扰防治登革病毒感染的进一步应用研究.
prM protein is a precursor of dengue virus membrane protein M. Membrane protein M plays an important role in virus assembly and maturation. Small interfering RNA (siRNA) designed for prM gene can inhibit dengue virus replication in short term. In this study, a recombinant lentivirus with prM siRNA sequence inserted was constructed. The flow cytometry (FCM) and blastomycin resistance were used to screen the Vero cells stably expressing prM siRNA. The results of RT-PCR and sequencing showed that the expression of prM siRNA in Vero cells was about 97.6% .Vero cells expressing prMsiRNA could significantly inhibit the expression of prM gene of dengue virus when challenged with dengue virus , And inhibit the replication of dengue virus in Vero cells.The established Vero cell line can be used for further application of RNA interference in prevention and treatment of dengue virus infection.