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目的 观察丙型肝炎病毒(HCV)RNA特异性脱氧核酶的体外剪切活性,探讨其用于抗病毒基因调控的可能性。 方法 以HCV 5’-非编码区及部分C区(5’-NCR-C)中两个具有5’…A ↓ U…3’特征的序列为靶位,以5’GGCTAGCTACAACGA3‘为活性中心,设计并合成两端经硫代修饰的脱氧核酶(TDRz),即TDRz-127和TDRz1。用Nar 1将质粒pHCV-neo完全线性化后,以之为模板体外转录获取HCV 5’-NCR-C;用碱性磷酸酶去除其5’端磷酸,再用T4聚核苷酸激酶和γ-32P-ATP进行5’端放射陛标记。在pH7.5、Mg2+10 mmol/L等条件下,将两种TDRz分别(5μmol/L)或共同(各2.5μmol/L)与底物RNA(200 nmol/L)混合,经变性、复性后于37℃孵育,分别于不同的时间点取出等份终止反应。以8％变性聚丙烯酰胺凝胶电泳和放射自显影分离、显示剪切产物,在凝胶成像分析仪上分析各条带光密度值并计算剪切百分率。 结果 在所设常用反应条件下于37℃孵育15、30、45、60、75、90min后,TDRz-127的剪切百分率分别达8.3％、16.1％、24.3％、26.2％、29.4％和31.1％,TDRzl的剪切百分率分别达7.4％,13.0％、15.6％、18.7％、19.4％和20.3％,两者同时剪切时的百分率约15.1％、29.6％、37.8％、39.1％、41.5％、42.6％。结论 TDRz-127和TDRzl对HCV 5’-NCR-C的剪切百分率随时
Objective To observe the in vitro cleavage activity of Hepatitis C virus (HCV) RNA-specific DNAzyme and to explore its potential for regulation of antiviral genes. Methods Two 5 ’... A ↓ U ... 3’ sequences of 5 ’non-coding region and part of C region (5’-NCR-C) were used as targets and 5’GGCTAGCTACAACGA3’ The thio-modified deoxyribozyme (TDRz) was designed and synthesized at both ends, namely TDRz-127 and TDRz1. After the plasmid pHCV-neo was completely linearized with Nar 1, HCV 5’-NCR-C was obtained by in vitro transcription. The 5’-NCR-C was removed by alkaline phosphatase and then treated with T4 polynucleotide kinase and γ -32P-ATP for 5 ’radiolabel. The two TDRz (5μmol / L) or common (2.5μmol / L) and substrate RNA (200 nmol / L) were mixed at pH7.5 and Mg2 + 10 mmol / After incubation at 37 ℃, respectively, at different points in time taken out of equal termination of the reaction. Separation with 8% denaturing polyacrylamide gel electrophoresis and autoradiography revealed the cleaved product, analyzed for the value of the optical density of each band on a gel imaging analyzer and calculated the percent shear. Results The percentage of cleavage of TDRz-127 reached 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1 respectively after being incubated at 37 ℃ for 15, 30, 45, 60, 75 and 90 min under the established reaction conditions % And TDRzl were 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3%, respectively. The percentages of simultaneous cleavage were 15.1%, 29.6%, 37.8%, 39.1% and 41.5% , 42.6%. Conclusions The percentage of cleavage of HCV 5’-NCR-C by TDRz-127 and TDRzl over time