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目的研究观察自行构建的siRNA表达载体对柯萨奇病毒B3(CVB3)感染的潜在治疗作用。方法以CVB3聚合酶编码区基因序列为靶点,构建发夹型siRNA真核表达载体pGCsi-U6/GFP/P及与CVB基因组序列无关的阴性对照pGCsi-U6/GFP/N。利用脂质体转染进入Hela细胞后,用CVB3毒株进行攻击,观察感染后Hela细胞病变情况,并用RT-PCR法检测细胞培养上清中的CVB3负链RNA水平。结果构建的表达质粒pGCsi-U6/GFP/P在Hela细胞能够正确表达,其表达产物能够使CVB3基因沉默,并间接抑制了CVB3的复制。结论本实验成功构建了能够抑制CVB3的发夹状siRNA表达质粒,该质粒具有增强细胞抗CVB3感染的能力。
Objective To investigate the potential therapeutic effect of self-constructed siRNA expression vector on Coxsackievirus B3 (CVB3) infection. Methods The hairpin siRNA eukaryotic expression vector pGCsi-U6 / GFP / P and the negative control pGCsi-U6 / GFP / N which was independent of the CVB genomic sequence were constructed. After transfected into Hela cells by liposome, the cells were challenged with CVB3 strain to observe the pathological changes of Hela cells after infection. The level of CVB3 negative strand RNA in cell culture supernatants was detected by RT-PCR. Results The constructed expression plasmid pGCsi-U6 / GFP / P was able to express correctly in Hela cells. The expressed product could silence CVB3 gene and indirectly inhibit the replication of CVB3. Conclusion The present study successfully constructed a hairpin siRNA expression plasmid capable of inhibiting CVB3, which has the ability to enhance the cell resistance to CVB3 infection.