| Project/Area Number |
63570212
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Virology
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| Research Institution | Kyushu University |
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Principal Investigator |
SATO Hiroyuki Kyushu University, Faculty of Medicine, Lecturer, 医学部, 講師 (70136456)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Human Parvovirus / Monoclonal Antibody / Neutralizing Antibody / Gene Expression / モノクローナル抗体 / ウイルスゲノムのクローニング |
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| Research Abstract |
Human parvovirus B19 can replicate only in erythroid precursors in bone marrow and fetal liver in vitro. This extremely dependence on host cell differentiation causes the limited materials of the virus and makes difficult the early diagnosis of the infection and vaccination for high risk group patient such as recipient of bone marrow transplantation, patients suffering from hemolytic anemias and pregnant women. In this study, we identified the site of viral capsid protein which involved the neutralization of the virus, using monoclonal antibody and site specific synthetic polypeptides, and also successfully observed the expression of VP-2 in mammalian cells and E.coli which could be used for the antigen for the serological diagnosis and vaccination as follow.
1.Identification of neutralization responsive site in VP-2 We obtained a monoclonal antibody BE11 which protected decrease of CFU-e in cultured human bone marrow cells challenged by the virus in vitro. The site of epitope of this monoclonal antibody was determined using 10 synthetic polypeptides deduced by the amino acid sequence of VP-2. BE11 only reacted a peptide which represented from 328th to 344th amino acid from the N-terminal of VP-2.
2.Gene expression of VP-2 in mammalian cells Accumulation of 58 kd of viral protein in the nucleus was observed in COS monkey cells transfected a plasmid containing SV40 late promoter and coding region of VP-2. This suggests the existence of nuclear localization signal in VP-2.
3.Gene expression of VP-2 in E.coli. A plasmid was constructed by cloning the coding region of VP-2 into the downstream of the coding region of signal peptide of outer membrane protein of E.coli. A 58 kd of protein having the antigenicity of VP-2 was successfully expressed in the periplasmic space of E.coli. We are now setting up the assay system for clinical diagnosis using this recombinant protein.
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