CAJ | 학술논문

目的　探讨丁型肝炎病毒(Hepatitis D virus, HDV)核酶用于抗丙型肝炎病毒(Hepatitis C virus，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HCV RNA的HDV核酶RzC1、RzC2和RzC3。体外转录获取含HCV RNA 5'-非编码区(5'-noncoding region,5'-NCR)及部分C区在内的底物RNA（HCV RNA 5'-NCR-C），并进行5'端放射性标记。在pH 7.5、37 ℃、Mg2+ 20 mmol/L和去离子甲酰胺2.5 mol/L等条件下，将核酶和底物按摩尔比100∶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，90 min时分别达24.9%、20.3%；未观察到RzC3有剪切活性。结论　经过结构优化的HDV基因组核酶在合适的位点能够剪切异源性RNA分子HCV RNA。
목적　탐토정형간염병독(Hepatitis D virus, HDV)핵매용우항병형간염병독(Hepatitis C virus，HCV)기인치료적가능성。방법　이HDV기인조핵매적가결양결구위기출，우화기경Ⅳ구，개건기저물결합구，획득3충침대HCV RNA적HDV핵매RzC1、RzC2화RzC3。체외전록획취함HCV RNA 5'-비편마구(5'-noncoding region,5'-NCR)급부분C구재내적저물RNA（HCV RNA 5'-NCR-C），병진행5'단방사성표기。재pH 7.5、37 ℃、Mg2+ 20 mmol/L화거리자갑선알2.5 mol/L등조건하，장핵매화저물안마이비100∶1혼합，재불동적시간점관찰전절백분솔。 결과　RzC1、RzC2대저물적전절백분솔수시간연장이체증，90 min시분별체24.9%、20.3%；미관찰도RzC3유전절활성。결론　경과결구우화적HDV기인조핵매재합괄적위점능구전절이원성RNA분자HCV RNA。
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 RzC1, RzC2 and RzC3 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, Mg2+ 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　RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 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.