2001 Fiscal Year Final Research Report Summary
Development of Replication System using A Hepatitis C Virus Infectious Clone and its Application
| Project/Area Number |
12670290
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Virology
|
|---|
| Research Institution | National Cancer Center Research Institute(C)(2) |
|---|
Principal Investigator |
SUGIYAMA Kazuo National Cancer Center Research Institure, Virology Division, Stuff, 研究所・ウイルス部, 研究員 (10242520)
|
|---|
| Project Period (FY) |
2000 – 2001
|
| Keywords | Hepatitis C virus / Replicon / MT-2C cell / Huh-7 cell |
|---|
| 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.
|
Research Products
(6 results)
