| Project/Area Number |
18590114
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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 |
Drug development chemistry
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| Research Institution | Kyushu University of Health and Welfare |
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Principal Investigator |
SADAKANE Yutaka Kyushu University of Health and Welfare, School of Pharmaceutical Sciences, Full-time instructor (60293304)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAHARA Masahiro Kyusyu University of Health Welfare, School of Pharmaceutical Sciences, Professor (40224828)
KONOHA Keiko Kyusyu University of Health Welfare, School of Pharmaceutical Sciences, Research associate (00369175)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,910,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | photoreaction / diazirine / antibody-displayed phage / amyloid / proteome / panning / screening / peptide polymerization |
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| Research Abstract |
We developed a method for an efficient cloning of display phages using photoreactive ligand, which was based on the combination of two heterogeneous techniques, photoaffinity labeling and panning of display phages. Photoaffinity labeling introduces a cross-link between a ligand and its specific binding proteins for fixing their affinity relationship through a covalent bond. We used 3-ary1-3-1rifluoromethyl-diazirine as a photophore because it appears to come closest to satisfy the criteria required for useful photophore. In our photochemical panning, the photoreactive ligand baring carbene-generating photophore, diazirine functions as a barb on a hook, onto which ligands are placed as fish bait Thus, the cross-linked phages stay on the ligand even after intensive washing, whereas almost all the unspecific binding phages are removed.
In this study, we developed the photoreactive panning method for screening of antibody-displayed phage, which was categorized in M13 filamentous phage. We succeeded the panning of antibody-displayed phage by using avidine-magnet beads and photoreactive antigen peptide, which had diazirine as a photophore, and biotin at N-terminal for a binding tag for avidine. Our developed panning methods enabled to clone the target displayed phage with higher sensitivity than the traditional panning did.
We found that a certain peptide fragment of prion protein inhibited the growth of rat primary culture cell of hippocannpus, and also that N-terminal structure of Alzheimer's beta-amyloid peptide altered spontaneously. We synthesized the peptide N-temiinal of which altered and tried to isolate the antibody-displayed phage that was reacted with such alternation. The synthesized peptide was immunized to five mice and their serums were obtained. We confirmed the production of antibody in the senims by ELISA with antigen.
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