| Project/Area Number |
17590612
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Gunma University |
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Principal Investigator |
SATO Ken Gunma University, Faculty of Medicine, Assistant, 医学部, 助手 (40396619)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | phage display / HCV core protein / molecular targeted drug / biopanning / shotgun scanning / phage library / bacteriophage / peptide / ファジーライブラリー |
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| Research Abstract |
We suubcloned DNA that codes HCV core protein (full-length) and truncated HCV core protein to GST-fusion vectors using HCV core protein expression vector to produce each HCV core protein. However the purification of HCV core proteins were difficult probably due to the hydrophobicity of C-terminus of HCV core protein even though we changed the design of the primers and the conditions of the purification of HCV core proteins. Therefore we purchased several commercially available more truncated HCV core proteins. The phage library expressing the numerous peptides, which I had been using in the lab in USA, was partly thawed and examined for the size of the diversity of the library. However the diversity was extremely decreased. Then we made the new library with l0^9+ copied of the diversity. We did biopanning by using a kind of HCV core protein. We got phages that could bind to GST protein nonspecifically or very weekly. We changed a HCV core protein to another kind of one and arranged the
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conditions of the experiments. However we could not get the phages that could bind enough specifically. Then we remade and redesigned the phage library and continued to arrange the experimental conditions such as washing and binding time. We found the candidates of clones with the binding ability, however, the specificity of these was considered to be insufficient by conducting reevaluation. We consulted with previous colleagues in the lab in USA and tried to redesign the library and arrange the experimental conditions again. However the detection of promising phages was difficult. Therefore we purchased the phage libraries that expressed commercially available circular and linear peptides. We also purchased the totally different phage display system in which foreign peptides was expressed on P3 viral coat proteins instead of P8 viral coat proteins. We are currently repeating biopanning by changing the experimental conditions.
On the other hand, we reported that zinc is a negative regulator of hepatitis C virus RNA replication using HCV replicon system expressing HCV core protein. We can evaluate the function of the target ligands binding to HCV core protein using this system in the future. Less
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