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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 19nt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VeroE6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。
Objective To construct specific siRNA plasmids against SARS CoV and use RNA interference to inhibit the replication and infection of the virus. Methods Based on the full-length cDNA sequence of SARS CoV, four oligonucleotides were designed and synthesized based on the full-length cDNA sequence of SARS-CoV. After annealed, oligonucleotides complementary to 19 nt were designed and synthesized The sequence was cloned into the pSilencer3.1 H1 vector to construct a plasmid expressing siRNA. The plasmids were transfected into VeroE6 cells by lipofectamine. After selection by hygromycin resistance, the stable expression of cytopathic effect, cytomegalovirus plaque formation and MTT assay were performed to observe the interference effect. Results The constructed plasmids were sequenced separately to confirm that the sequences containing siRNAs were consistent with the expected specific interference sequences. Cells transfected with different concentrations of SARS CoV plasmid showed reduced cytopathicity, a significant increase in viable cells, and a significant reduction in viral plaques compared with the negative control. Conclusion The RNA interference can effectively inhibit the replication of SARS CoV in cells and protect the cells against SARS. It provides new ideas and methods for the prevention and treatment of SARS.