| 研究課題/領域番号 |
18K04905
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| 研究機関 | 北陸先端科学技術大学院大学 |
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研究代表者 |
ビヤニ マニッシュ 北陸先端科学技術大学院大学, エクセレントコア推進本部, 特任教授 (00599780)
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| 研究分担者 |
高村 禅 北陸先端科学技術大学院大学, 先端科学技術研究科, 教授 (20290877)
山下 啓子 北海道大学, 大学病院, 教授 (70332947) [辞退]
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| 研究期間 (年度) |
2018-04-01 – 2022-03-31
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| キーワード | Peptide aptamer / Voltammetric sensor / Brest Cancer / Cathepsin E |
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| 研究実績の概要 |
Two research themes were followed:
(Research 1) Immobilization of functional peptide aptamer on carrier magnetic microbeads: First, a biotinylated alpha-helix inducing ligand is utilized to immobilize the CatE-aptamer peptide onto a carrier bead, i.e., streptavidin coated magnetic beads. Second, a pentapeptide ligand coupled with alpha-helix inducing ligand is utilized to immobilize the CatE-substrate peptide onto gold nanoparticles. The terminal Cysteine of pentapeptide is coupled with BSA protein via NHS-ester of BSA protein and pyridyldithiol reactive group of SPDP crosslinker. The complex is purified using gel filtration column and confirmed by SDS-PAGE analysis. Next, the CatE-substrate peptide coupled BSA is physically adsorbed onto the gold nanoparticles. The binding efficiency of peptide on nanoparticles is confirmed by lateral flow assay.
(Research 2) Binding, activity assay and sensitivity evaluation of CatE in a known sample solution: The binding of CatE-aptamer peptide with CatE enzyme which is bounded on magnetic beads are performed. The protease activity assay of CatE enzyme to cleave CatE-substrate peptide is performed at neutral pH. The protease activity assay is evaluated by electrochemical measurement. The peak height which represents the cleaved part of CatE-substrate peptide increased with the concentration of CatE. In next, the activity assay of CatE is evaluated in the presence of CatE-aptamer peptide. A 0.01 nmol concentration of CatE-aptamer peptide is optimized with higher current signals.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
The newly designed peptide which distinctly cleaved at neutral pH with special preference for Arg-Arg was synthesized and confirmed. However, a joint collaborative work with co-applicant (山下先生, 北海道大) could not be perform due to the sudden retirement of co-applicant from the project.
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| 今後の研究の推進方策 |
We are looking forward to find a possible collaborator as an alternative of retired co-applicant (山下先生, 北海道大). We expect to receive and analyze clinical samples to validate the newly designed peptide.
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| 次年度使用額が生じた理由 |
The unused budget in FY2020 was mainly due to the no progress of clinical sample analysis. This is planned to be continued in FY 2021.
1) Fee to hire a researcher to perform experiments; 2) Cost for reagents and performing experiments 3) Travel cost to make a presentation of project work in one international conference
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