【作者】 连建奇；

【导师】 周永兴； 金由辛；

【作者基本信息】 第四军医大学 ， 传染病学， 1997， 博士

【摘要】 乙型肝炎其病原体是乙型肝炎病毒(Hepatitis B virus，HBV)。HBV是一小的DNA病毒，其复制过程需要经过RNA的生物合成阶段。HBV C基因表达的C蛋白(HBcAg、HBeAg)参与病毒核衣壳的装配，是免疫攻击的靶抗原，因此剪切C基因mRNA既可抑制HBV的复制，又可阻断HBcAg的表达。由于锤头型核酶具有分子小，可序列特异性地切割靶RNA的特点，所以可用它切割HBV C基因的RNA，阻断HBV基因的复制和表达。本研究设计多位点核酶观察其对靶RNA的切割作用。 利用计算机对HBV C基因的mRNA二级结构进行分析，设计并合成了5’顺式、3’顺式核酶及针对C基因RNA的三个锤头状核酶基因，三个核酶分别剪切C基因mRNA上的不同位点。将上述核酶基因构建成带有5’和3’顺式核酶的体外转录质粒中，其中包括单个、两个或三个串联的针对靶基因的核酶基因质粒。体外转录结果表明我们构更多还原

【Abstract】 Hepatitis B virus(HBV) , the causative agent of B-type hepatitis in human, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. The core protein , the most important component in formation of HBV nucleocapsid, is closely related to the replication of HBV DNA. Cleavage of the pregenomic RNA and/or mRNA of core gene, therefore, may interrupt the life cycle of HBV. Hammerhead ribozymes are short catalytic RNA molecules possessing endoun-clease activity. The enzymes specificity is mediated by variable flanking sepuences complementary to the RNA target substrate. In this report, we describe multitarget ribozymes construct that cleave a critical region of the HBV pregenomic RNA and the ribozymes expression in the HepG2 cells.By computer program, we first analysed the secondary structure of target RNA, and designed two cis —ribozymes (intramolecularly) and three trans —ribozymes (intermolecularly), which each one is specific for a different cleavage site on the mRNA of core gene. Then we constructed plasmids combining 5’ and 3’ trimming system (two cis—ribozymes) with each ribozyme gene or connected -type ribozyme genes containing different two ribozyme genes and a triple ribozyme gene. These plasmids were designed to release antiviral molecules (trans更多还原