| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
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.
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