| Project/Area Number |
02454097
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
基礎獣医学
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
SHINAGAWA Morikazu Obihiro Univ., Dept. of Vet. Public Health, Professor, 畜産学部, 教授 (00001537)
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| Co-Investigator(Kenkyū-buntansha) |
HORIUCHI Motohiro Obihiro Univ., Dept. of Vet. Public Health, Assistant, 畜産学部, 助手 (30219216)
ISHIGURO Naotaka Obihiro Univ., Dept. of Vet. Public Health, Asso. Prof., 畜産学部, 助教授 (00109521)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1991: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1990: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | Scrapie / PrP gene / Cell culture / Gene expression / spongiform encephaloparthy / 分子クロ-ニング |
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| Research Abstract |
A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and 6 amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences like other animal PrP genes and consisted of 256 amino acids, the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to the 165th amino acid from the N-terminal. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP^c. The reoombinant PrP^c co-migrated with authentic bovine PrP^c in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at perinuclear region of the cells.
Subtractions, a nucleic acid fraction and a PrP fraction consisting of PrP^<17-25>. a core fragment of PrP^<sc>, were prepared from the mouse scrapie-associated fibril-enriched fraction. To examine the biological activity, the nucleic acid fraction was either first introduced into mouse L-929 cells before or after nuclease treatments, then transfected cell lysates prepared 2 weeks later were inoculated into mice, or directly inoculated into mice with or without the PrP fraction. The PrP fraction alone was also inoculated into mice. Mice inoculated with the transfected cell lysates or with the nucleic acid fraction alone showed no scrapie signs during their lifespan. While 60% of the mice inoculated with the PrP fraction alone and 67% of those inoculated with the fraction together with the nucleic acid fraction showed clinical signs of scrapie.
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