| Project/Area Number |
06404029
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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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 | University of Tokyo |
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Principal Investigator |
OMATA Masao University of Tokyo, Faculty of Medicine, Professor, 医学部・附属病院, 教授 (90125914)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Naoya University of Tokyo, Faculty of Medicine, Staff, 医学部・附属病院, 医員
MATSUMURA Masayuki Asahi Life Foundation Institute for Adult Diseases Department of Gastroenterolog, 消化器科, 部長
SHIRATORI Yasushi University of Tokyo, Faculty of Medicine, Associate Professor, 医学部・附属病院, 講師 (70196624)
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| Project Period (FY) |
1994 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥24,800,000 (Direct Cost: ¥24,800,000)
Fiscal Year 1997: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1996: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1995: ¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1994: ¥7,300,000 (Direct Cost: ¥7,300,000)
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| Keywords | hepatitis B virus (HBV) / HLA / cytotoxic T cell / T cell receptor / YMDD motif / hepatitis C virus (HCV) / NS5A / transcriptional activator / HLA / 細胞障害性T細胞 / T細胞受容体 / B型肝炎 / 細胞障害性T細胞(CTL) / Core / エピトープ / T細胞受容体(TCR) / レパートリー / long-PCR / transfection / class I HLA / ウイルス変異 / HLA class I / Hep G2 / LC / MS / ペプタイドの構造解析 |
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| Research Abstract |
Hepatitis B virus (HBV)
We purified HLA-binding peptides from HBV stably-transfected human hepatoma cells (HepG2 2.2.15) by affinity chromatography using monoclonal Ab. Amino acid sequence of HLA-binding peptide was determined as NPLPOLPO by HPLC-mass spectrometry (LC/MS/MS).
Full-length HBV DNAs obtained from sera of patients with type B-chronic liver diseases were sequenced Amino acid substitutions were accumulated in core-region and pre-S region according to the progression of chronic liver disease.
The repertoire of T cell receptor alpha-chain of liver-invaded cytotoxic T cells in patiens with type B-chronic active hepatitis, was determined by inverse PCR,cloning and sequencing. The Valpha 7.2 gene was most frequently observed and found to bind Jalpha 33 gene.
We established one-step amplification of 3.2 kbp of full-length HBV DNA by using long-PCR method. Cloned full-length HBV DNA was confirmed to replicate by transfection into human hepatoma cells (Huh7). We made 7 variants by substituting the methionine of the YMDD motif with Ile, Val, Ala, Leu, Lys, Arg and Thr, to explore replication ability and lamivudine sensitivity of these variants. Four variants (Ile, Val, Ala and Leu) remained replication competent, whereas 3 others (Lys, Arg and Thr) showed impaired replication. 2 variants (Ile and Val) were showed to be resistant to lamivudine.
Hepatitis C virus (HCV)
Hepatitis C virus nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids fused to the DNA-binding domain of GAL4 strongly activates transcription in yeast and human hepatoma cells (Huh7). Transcriptional activation by HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis.
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