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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
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 )
