目的：寻找高效特异的新型抗HCV药物, 探讨针对HCV基因的硫代修饰反义寡核甘酸（S-ASODNs）最佳作用靶序列。 方法：参考HCV 5′NCR计算机预测的二级结构图，设计合成了15条S-ASODN、3条正义寡核苷酸及1条随机序列，采用HCV 5′NCR调控荧光素酶基因的瞬时表达系统，将S-ASODN与pHCV-neo4共转染，通过荧光素酶活性表达高低，反映S-ASODN对HCV调控基因的抑制活性。结果：5条S-ASODN即HCV65、HCV279、HCV363、HCV349及HCV352在25～100nmol/L浓度范围内，对HCV调控基因具有特异性的剂量依赖性抑制活性。当浓度为100nmol/L时，这些ASODN的抑制率可达80％以上，IC50值提示HCV363的抑制活性最强。通过阴性对照实验包括正义寡核苷酸、随机序列及不含HCV序列的靶载体pGL3提示ASODN仅存在轻度非特异性作用。此外，实验结果还提示不同作用靶位的ASODN之间具有一定的协同作用。 结论：HCV 5′NCR第二茎环结构Ⅱa、第三茎环结构Ⅲd和翻译起始区可能是ASODN作用的重要靶序列。%Objective: To screen efficient and specific new drugs against hepatitis C virus (HCV) and find the best target sequence of antisense oligodeoxynucleotides (ASODNs) targeting at HCV gene. Methods: Fifteen S-ASODNs targeting at the 5'NCR and start AUG of HCV RNA were designed and synthesized according to the predicted RNA secondary structure of the HCV 5'NCR and the adjacent AUG region. HepG2 cells were co-transfected with pHCV-neo4 and S-ASODN. The inhibitory effects of S-ASODNs on gene expression controlled by HCV 5'NCR were determined by the assay of luciferase activity. Results: Five S-ASODNs, i.e. HCV65, HCV279, HCV363, HCV349 and HCV352, showed sequence-specific and dose-dependent inhibitory activities with an inhibition rate of more than 80% at a concentration of 100 nmol/L. According to 50% inhibitory concentrations, the inhibitory activity of HCV363 was the best. On the other hand, ASODNs had only little non-specific effect by negative control trials. In addition, the results also indicated that there were some coordinate effects between ASODNs at different targets. Conclusion: Stem-loop Ⅱa and Ⅲd of HCV 5'NCR and the start AUG of polyprotein precursor are candidate targets of S-ASODNs.
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