Development of Replication System using A Hepatitis C Virus Infectious Clone and its Application

丙型肝炎病毒感染性克隆复制系统的研制及其应用

• 批准号: 12670290
• 负责人: SUGIYAMA Kazuo
• 金额: $ 0.64万
• 依托单位: National Cancer Center Research Institute(C)(2)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670290/
• 关键词: Hepatitis C virus; Replicon; MT-2C cell; Huh-7 cell; MT2-C細胞; 感染性クローン; レトロウイルスベクター

We have been attempting to obtain, from a Hepatitis C virus (HCV)-infected MT-2c cells, a HCV clone which is capable of replication. We amplified two fragments, namely the structure and non-structure regions, and cloned them into a full-length genome. In this study, we attempted to establish subgenomic HCV RNA replicon bearing a consensus sequence.We inserted a neomycin resistant gene into the full-length genome bearing a consensus sequence in the place of the structure region, and created a replicon expressing plasmid. Huh-7 cells were transfected with RNA synthesized in vitro using this plasmid and cultured in the presence of G418.As a result, some colonies were observed and cloned. Subgenomic HCV RNA was detected in the clone, and de novo synthesis of subgenomic HCV RNA was also detected. In addition, non-structure proteins of HCV were detected by Western blotting. Taken together, subgenomic HCV RNA generated in the present study served as RNA replicon, which is capable of self-replication.Then we developed a method to generate a replicon library form various HCV sources. We are on the process of establishment of replicon library from type C hepatitis patients. And also, we are planning to develop a transient system using this replicon to screen anti-HCV drugs.

我们一直试图从丙型肝炎病毒（HCV）感染的MT-2c细胞中获得能够复制的HCV克隆。我们扩增了两个片段，即结构区和非结构区，并将它们克隆成全长基因组。在本研究中，我们试图建立具有共有序列的亚基因组HCV RNA复制子。我们将新霉素抗性基因插入到具有共有序列的全长基因组的结构区域中，并创建了复制子表达质粒。使用该质粒用体外合成的RNA转染Huh-7细胞，并在G418存在下培养。结果，观察到并克隆了一些集落。在克隆中检测到亚基因组HCV RNA，并且还检测到亚基因组HCV RNA的从头合成。此外，通过Western blotting检测HCV的非结构蛋白。总而言之，本研究中产生的亚基因组HCV RNA作为RNA复制子，能够自我复制。然后我们开发了一种从各种HCV来源生成复制子库的方法。我们正在建立丙型肝炎患者的复制子库。此外，我们还计划开发一个使用该复制子的瞬态系统来筛选抗丙肝病毒药物。

项目成果

Naganuma A, Sugiyama K: "Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein"J Viral. 74. 8744-8750 (2000)

Naganuma A、Sugiyama K：“丙型肝炎病毒核心蛋白激活干扰素诱导型 2-5-寡腺苷酸合成酶基因”J Viral。

T.Tanaka,K.Sugiyama: "Hepatitis C virus NS5B RNA replicase specifically binds ribosomes."Microbiol.Immunol.. 44. 543-550 (2000)

T.Tanaka,K.Sugiyama：“丙型肝炎病毒 NS5B RNA 复制酶特异性结合核糖体。”Microbiol.Immunol.. 44. 543-550 (2000)

Naganuma A, Sugiyama K: "Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein"J Virol. 74. 8744-8750 (2000)

Naganuma A、Sugiyama K：“丙型肝炎病毒核心蛋白激活干扰素诱导型 2-5-寡腺苷酸合成酶基因”J Virol。

Ikeda M, Suglyama K: "Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells"Virus Res. 66. 51-63 (2000)

Ikeda M、Suglyama K：“乳铁蛋白对人类培养细胞中丙型肝炎病毒感染的抗病毒活性的表征”Virus Res。

Ikeda M, Sugiyama K: "Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells"Virus Res. 66. 51-63 (2000)

Ikeda M，Sugiyama K：“乳铁蛋白对人类培养细胞中丙型肝炎病毒感染的抗病毒活性的表征”Virus Res。