| Project/Area Number |
11670478
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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 |
MITUSI Hiroshi Gastioenterology, The University of Tokyo, assistant professor, 医学部・附属病院, 助手 (30239280)
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| Co-Investigator(Kenkyū-buntansha) |
MAEKAWA Hisato University Hospital, Gastioenterology, The University of Tokyo, professor, 医学部・附属病院, 助手 (10301102)
MARUYAMA Toshiyuki Faculty of Medicine, The University of Tokyo, lecturer, 大学院・医学系研究科, 講師 (30219571)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | hepatitis C virus / viral receptor / C型肝炎 / レセプター |
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| Research Abstract |
We tried to express hepatitis c virus (HCV) envelope proteins : E1 and E2 in mammalian cells and to purify them partially. First, RNA was extracted from patient serum including genotype Ib virus. Then, cDNA was synthesized and E1 and E2 DNA were amplified by PCR using specific primers with 6 His-tag. They were constructed into an expression vector and sequenced. COS7 cells were transected with the plasmids and expression of the envelope proteins were detected by Western blotting. Among the envelope proteins espressed, only C-terminal deleted E2 protein (to amino acid 661) was secreted into the culture medium and could be purified partially by Ni-NTA column. We are now trying to screen a human liver library with the purified protein in a expression cloning method. In addition, we cloned human CD81 which was reported to bind HCV E2 by PCR. COS7 cells were trasnfected with the plasmid containing CD81, and the expression was detected by FACscan and immunocytochemistry. CD81-expressed COS7 cells were incubated with patient serum and samples were drawn periodically. So far minus strand of HCV was not detected in cells or supernatants. The data indicated that only CD81 protein is not enough for HCV to enter and amplify in mammalian cells.
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