质粒表达型siRNA对SARS-CoV复制与感染干扰的初步研究

目的构建针对SARS-CoV的特异性siRNA质粒,利用RNA干扰技术抑制该病毒复制与感染.方法根据SARS-CoV的全长基因序列,以4个不同的部位为靶点,分别设计、合成含有19 nt干扰序列的一段寡核苷酸,将两两互补的寡核苷酸经退火后所形成的序列克隆到pSilencer 3.1-H1载体中,构建表达siRNA的质粒.采用脂质体法将质粒转染Vero E6细胞,经潮霉素抗性筛选后,再对稳定表达的细胞进行细胞病变、病毒空斑形成和MTT实验,以观察干扰效应.结果对所构建的质粒分别测序,确定其含有siRNA的序列与预期的特异性干扰序列一致.用不同浓度的SARS-CoV感染转染质粒后的细胞,与阴性对照相比,其细胞病变减轻、活细胞显著增加,病毒空斑明显减少.结论利用RNA干扰能有效的抑制SARS-CoV在细胞中的复制,并对细胞有保护作用,为预防、治疗SARS提供了新的思路和方法.

Inhibition of severe acute respiratory syndrome-associated coronavirus replication and infection by plasmid-mediated siRNA

Objective To inhibit severe acute respiratory syndrome-associated coronavirus (SARS-CoV) replication by constructing specific small interfering RNA (siRNA) vectors which can induce RNA interference (RNAi). Methods The hairpin siRNA template oligonucleotides that target SARS-CoV were designed and synthesized according to the whole genomic sequence of this virus. The products of annealing the hairpin siRNA template oligonucleotides respectively were cloned into vector pSilencer 3.1-H1 to construct the plasmids expressing siRNA. After these plasmids were transferred into Vero E6 cells by DOTAP reagent, the stable cells expressing short hairpin siRNA were obtained by using hygromycin selection. Then CPE and MTT experiments and plaque reduction array were conducted with above cells. Results The sequencing results indicated that the sequences cloned into vectors were identical to the expected. Combined with the negative control, the number of alive cells transfected plasmids expressing siRNA increased dramatically under SARS-CoV attack. Conclusion The inhibitive effect of RNAi on SARS-CoV replication may provide us a new strategy to prevent and treat SARS.