表达登革病毒prM基因小干扰RNA的Vero细胞系的建立

Establishment of Vero Cell Lines with Expression of Small Interfering RNA Targeting Dengue Virus prM Gene

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 is the precursor of a membrane protein M that plays an important role in the assembling and maturation of dengue virus.Small interfering RNA（siRNA） targeted to prM gene can transiently inhibit the replication of dengue viruses.In order to establish persistent inhibitory effect,recombinant lentiviruses with prM siRNA sequence were constructed and produced in 293FT cells,then transfected into African green monkey kidney cells（Vero cells）.Vero cells stably expressing of prM siRNA were selected by blasticitin resistance and fluorescence activating cell sorter（FACS） sorting.The sequence of expressed siRNA was confirmed by reverse transcript PCR and DNA sequencing.The prM siRNA expression was detected in about 97.6% of the obtained Vero cells.When exposed to dengue viruses,prM siRNA expression Vero cells showed significant suppression on prM protein expression,and inhibition on the replication of dengue viruses.These cell lines can be used for future studies of RNA interference on the protection of dengue virus infection.