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目的 研究特异性脱氧核酶对丙型肝炎病毒(HCV)的体外消化作用。方法 设计3个分别 作用于 HCV RNA 5′-非编码区(5′-NCR)上157、168、173位点的脱氧核酶(DRz)，分别命名为 DRzl， DRz2，DRz3，均以5′-GGCTAGCTACAAcGA-3′为脱氧核酶的催化活性中心。用内切酶 Xba Ⅰ将含有 HCV 病毒序列的质粒 pCMV/T7-NCRC-△Luc 消化成线性，以此为模板体外转录出用[α-~(32)p]UTP 进行标 记的靶 HCV RNA。在37℃、pH 7．5、Mg~(2+)10 mmol/L 等条件下，将 HCV RNA(10 nmol/L)与3个脱 氧核酶(1 μmol/L)分别混合进行切割反应，并进一步比较不同 Mg~(2+)浓度下 DRz3的切割反应效率，反应 产物在8%变性聚丙烯酰胺凝胶上电泳分离并行放射自显影，应用凝胶成像分析仪评价脱氧核酶的切割效率。 结果 在设定反应条件下，3个脱氧核酶对 HCV 病毒均有切割活性。随着 Mg~(2+)浓度的增加，DRz3的切割 活性增强。结论 特异性的脱氧核酶可有效切割 HCV RNA 5′NCR 的 RNA，且切割效率与 Mg~(2+)浓度呈 正相关。
Objective To study the in vitro digestion of hepatitis C virus (HCV) by specific DNAzyme. Methods Three deoxyribozymes (DRz) acting on 157,168,173 sites of 5’-NCR in HCV RNA were designed and named as DRzl, DRz2 and DRz3, -GGCTAGCTACAAcGA-3 ’is a catalytic activity center of DNAzyme. The plasmid pCMV / T7-NCRC-Δ Luc, containing the HCV viral sequence, was digested to linear with the endonuclease Xba I and used as a template to transcribe the target HCV RNA labeled with [α- (32) p] UTP in vitro. HCV RNA (10 nmol / L) and 3 DNAzyme (1 μmol / L) were mixed and cut at 37 ℃, pH 7.5, Mg 2+ 10 mmol / Furthermore, the efficiency of cleavage reaction of DRz3 at different concentrations of Mg 2+ was further compared. The reaction products were electrophoretically separated by 8% denaturing polyacrylamide gel and autoradiographed. The gel imaging analyzer was used to evaluate the cleavage efficiency of DNAzyme. . Results Under the set reaction conditions, all 3 deoxyribozymes had cleavage activity on HCV. With the increase of Mg 2+ concentration, the cleavage activity of DRz3 increased. Conclusion The specific deoxyribozyme can effectively cut RNA of 5’NCR of HCV RNA, and the cleavage efficiency is positively correlated with the concentration of Mg 2+.