锤头状核酶在HepG2细胞中抑制adr亚型的乙型肝炎病毒(英文)  

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells

作　　者：郑文超[1] 陆长德[1] 孔玉英[1] 汪垣[1] 祁国荣[1] 

机构地区：[1]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第1期25-29,共5页

基　　金：国家 8 6 3高技术研究和发展计划资助项目!(86 3 10 2 18 47);国家自然科学基金会!(No .2 96 32 0 6 0 )资助项目&&

摘　　要：针对HBV基因组设计了三种锤头状核酶———RS3、RC2和RC1。将裸露的和用tRNA包埋的核酶 (RtS3、RtC2和RtC1)基因插入RNA修剪质粒pRG5 2 3中 ,然后转变为真核表达载体。使用同样克隆技术 ,构建由不同长度 (2、4、8、12个 )RS3或 12个RtS3连成的鸟枪型真核表达载体。将它们分别与带有HBV基因组的质粒共转染人肝癌细胞系HepG2 ,用G418筛选抗性细胞。分析不同种类的核酶在G418抗性细胞中的HBV抑制活性 ,包括子代HBV DNA、HBV RNA和抗原 (HBsAg或HBcAg/HBeAg)表达的减少。结果表明 ,所有设计的核酶对HBV有 >70 %的抑制活性 ,而tRNA包埋核酶的活性略高于裸露核酶。特别值得提出的是 ,由 8个或 12个相同核酶连接的鸟枪型质粒 (8RS3、12RS3和 12RtS3)具有很高的抑制HBV活性 ,达到 >90Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun type plasmids carrying homogeneous RS3 or RtS3 unit connected in tandem were obtained. After co transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV inhibiting activity of different kinds of ribozyme in G418 resistant cells was achieved by measuring the decrease of HBV RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

关 键 词：锤头状核酶 乙型肝炎病毒 HBV HEPG2细胞 

分 类 号：Q789[生物学—分子生物学]