剪切HCV RNA的HDV核酶的设计及活性测定  

Design and activity assessment of HDV ribozymes to trans-cleave HCV RNA

作　　者：于乐成[1] 顾长海[1] 毛青[1] 李奇芬[1] 王宇明[1] 郭焕珍[1] 

机构地区：[1]第三军医大学附属西南医院全军感染病研究所,重庆400038

出　　处：《第三军医大学学报》2001年第3期312-314,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 !(3 960 0 0 3 1 )

摘　　要：目的　探讨丁型肝炎病毒 (HepatitisDvirus ,HDV)核酶用于抗丙型肝炎病毒 (HepatitisCvirus ,HCV)基因治疗的可能性。方法　以HDV基因组核酶的假结样结构为基础 ,优化其茎Ⅳ区 ,改建其底物结合区 ,获得 3种针对HCVRNA的HDV核酶RzC1、RzC2 和RzC3 。体外转录获取含HCVRNA 5’ 非编码区 ( 5’ noncodingregion ,5’ NCR)及部分C区在内的底物RNA(HCVRNA 5’ NCR C) ,并进行 5’端放射性标记。在pH 7.5、37℃、Mg2 + 2 0mmol/L和去离子甲酰胺 2 .5mol/L等条件下 ,将核酶和底物按摩尔比 1 0 0∶1混合 ,在不同的时间点观察剪切百分率。结果　RzC1、RzC2 对底物的剪切百分率随时间延长而递增 ,90min时分别达 2 4.9%、2 0 .3% ;未观察到RzC3 有剪切活性。Objective To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti hepatitis C virus (HCV) gene thera py drugs. Methods The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate binding region reconstructed, thus three recombinant HCV specific HDV genomic ribozymes RzC 1, RzC 2 and RzC 3 were obtained. HCV RNA 5' noncoding region and 5' fragment of C region (HCV RNA5' NCR C) were transcribed from plasmid pHCV neo by T7 phage RNA polymerase in vitro , and radiolabelled at its 5' end. The trans cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg 2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results RzC 1, RzC 2 trans cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac ting for 90 minutes respectively; RzC 3 was not able to trans cleave its substrate. Conclusion Recombinant HDV genomic ribozymes have the ability to trans cleave HCV RNA, but the appropriate target sequence should be selected.

关 键 词：丁型肝炎病毒 核酮 基因组 丙型肝炎病毒 基因治疗 剪切活性 HCV-RNA 

分 类 号：R512.630.5[医药卫生—内科学]