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

我们一直在尝试从丙型肝炎病毒（HCV）感染的MT-2c细胞中获得能够复制的丙型肝炎病毒克隆。我们扩增了两个片段，即结构区和非结构区，并将它们克隆成一个全长基因组。在这项研究中，我们试图建立具有一致序列的HCV RNA亚基因组复制子。我们将一个新霉素耐药基因插入到全长基因组中，在结构区有一个一致的序列，并创建了一个复制子表达质粒。用该质粒体外合成的RNA转染Huh-7细胞，并在G418存在下培养。结果，对一些菌落进行了观察和克隆。在克隆中检测到亚基因组HCV RNA，并检测到亚基因组HCV RNA的从头合成。Western blotting检测HCV非结构蛋白。综上所述，本研究中产生的HCV亚基因组RNA作为RNA复制子，具有自我复制的能力。然后，我们开发了一种从各种HCV源生成复制子库的方法。我们正在进行丙型肝炎复制子文库的建立。同时，我们正计划开发一种瞬时系统，利用这种复制子来筛选抗hcv药物。

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.