权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of anti-HCV drugs that inhibit HCV serine protease

开发抑制HCV丝氨酸蛋白酶的抗HCV药物

基本信息

批准号：
04557125
负责人：
MIYAMURA Tatsuo
金额：
$ 8.96万
依托单位：
National Institute of Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557125/
关键词：
HCV Serine protease Anti-viral drug Baculovirus プロテアーゼ分子生物学

项目摘要

HCV contains a positive-stranded RNA genome with approximately 9,400 bases. It comprises a single, large open reading frame encoding a polyprotein of 3010-3033 amino acids, and it appears to be most closely related to flaviviruses and pestiviruses on the basis of similarities of its genomic structure and organization. Recent investigations revealed that four cleavages on the HCV precursor polyprotein are mediated by a viral serine-type proteinase lying within the amino-terminal half of the NS3 protein. This proteinase is thought to be a target for antiviral agents, and experimental systems of the enzyme activity will lead to testing therapeutic agents that might specifically inhibit HCV polyprotein processing. Our results are summarized as follows ;(1) HCV NS3 serine proteinase was expressed in recombinant E.coli and baculovirus expression system, and the expressed protein exhibited specific proteolytic activity. (2) The enzyme was expressed in E.coli with His-Tag sequence at N-terminus and effectively purified with metal chelation resin. (3) High level expression of this enzyme was accomplished in baculovirus expression system by introducing rabies virus G protein signal sequence. (4) A human hepatoma cell line (HepG2) constitutively expressing proteins of HCV was established by transfection with HCV cDNA non-structural region. (5) A series of known proteinase inhibitors were tested by in vivo coexpression system and inhibition of the enzyme by TLCK was detected.

丙型肝炎病毒包含一个正链RNA基因组，大约有9,400个碱基。它由一个编码3010-3033个氨基酸的多蛋白的单一大的开放阅读框架组成，根据其基因组结构和组织的相似性，它似乎与黄病毒和鼠疫病毒关系最密切。最近的研究表明，丙型肝炎病毒前体多聚蛋白上的四种裂解是由位于NS3蛋白氨基末端的一种病毒丝氨酸蛋白酶介导的。这种蛋白水解酶被认为是抗病毒药物的靶点，这种酶活性的实验系统将导致测试可能特异性地抑制丙型肝炎病毒多蛋白处理的治疗剂。主要研究结果如下：(1)在重组大肠杆菌和杆状病毒表达系统中表达了丙型肝炎病毒NS3丝氨酸蛋白酶，表达的蛋白具有特异的蛋白降解活性。(2)该酶在大肠杆菌中得到高效表达，N端有His-Tag序列，金属螯合树脂纯化效果较好。(3)通过引入狂犬病病毒G蛋白信号序列，在杆状病毒表达系统中实现了该酶的高效表达。(4)将丙型肝炎病毒非结构区基因转入人肝癌细胞系(HepG2)，建立了表达丙型肝炎病毒蛋白的人肝癌细胞系(HepG2)。(5)用体内共表达系统检测一系列已知的蛋白水解酶抑制剂，并检测TLCK对该酶的抑制作用。