| Project/Area Number |
15590626
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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 | The University of Tokyo |
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Principal Investigator |
KANAI Fumihiko The University of Tokyo, Faculty of Medicine, Visiting Assistant Professor, 医学部附属病院, 客員助教授 (70334399)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | hepatitis C virus / NS3 / baculovirus / random peptide library / protease inhibitor / hepatocellular carcinoma / ペプチドライブラリー |
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| Research Abstract |
HCV-NS3 encodes a protease which cleaves its own translated peptide, and being considered one of the therapeutic target of this virus. Baculovirus expression provides correct folding of recombinant protein as well as disulfide bond formation, oligomerization and other important post-translational modifications. Consequently the overexpressed protein exhibits the proper biological activity and function. HCV-NS3 gene was amplified by PCR using pUC3241 (gift from Dr. Tetsuro Suzuki). The PCR fragment was subcloned into Spe I-Nde I site of pACGHLT-A(BD Biosciences Pharmingen). Viral solution containing virus expressing NS3 was prepared by transfection of plasmid into Sf9 insect cells using Bac-To-Bac system(Invitrogen). Sf9 insect cells were recovered after infection of GST-NS3 expressing virus. Cells were lysed and the supernatant was recovered by ultra-centrifugation. Glutathione Sepharose beads were added to the GST-NS3 lysate and incubated for 3 hours at 4℃. After washing beads, recombinant GST-NS3 protein was eluted and subjected to SDS-PAGE and staining. The molecular weight of the recombinant GST-NS3 protein was approximately 43kDa, and cross reacts with anti-NS3 antibody by western blotting. We obtained recombinant HCV-NS3 protein by baculovirus. In order to develop anti-NS3 protease drug, it is important to know the substrate specificity of the NS3 protease. One approach would be the use of random peptide library with recombinant NS3
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