| 研究課題/領域番号 |
20K16246
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| 研究種目 |
若手研究
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| 配分区分 | 基金 |
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| 審査区分 |
小区分49050:細菌学関連
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| 研究機関 | 大阪公立大学 (2022-2023)
大阪府立大学 (2020-2021) |
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研究代表者 |
アワスティ シャルダ 大阪公立大学, 大学院獣医学研究科, 特任講師 (30751888)
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| 研究期間 (年度) |
2020-04-01 – 2024-03-31
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| 研究課題ステータス |
完了 (2023年度)
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| 配分額 *注記 |
4,030千円 (直接経費: 3,100千円、間接経費: 930千円)
2022年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
2021年度: 1,300千円 (直接経費: 1,000千円、間接経費: 300千円)
2020年度: 1,300千円 (直接経費: 1,000千円、間接経費: 300千円)
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| キーワード | Vibrio cholerae / Cholix toxin / コレラ菌 / Receptor |
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| 研究開始時の研究の概要 |
Cholix toxin (ChxA) is a recently identified cytotoxin produced by Vibrio cholerae. The cytotoxic pathway and mouse lethality caused by liver injury has been gradually understood, however, the cell surface receptors are not yet known, which are necessary molecules for ChxA binding to target cells. My in silico analysis identified a potential toxin receptor. This study will evaluate my hypothesis by comparing toxin sensitivity of several cell lines expressing and non-expressing the candidate receptor molecule. In vitro binding assay and co-crystallization analysis will be also carried out.
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| 研究実績の概要 |
Cholix toxin (ChxA) is a recently identified cytotoxin produced by pathogenic Vibrio cholerae non-O1/non-O139. The cytotoxic pathway and mouse lethality caused by liver injury has been gradually understood, however, the cell surface receptors are not yet known, which are necessary molecules for ChxA binding to target cells. The objectives for the fiscal year 2023-24 were focused on attempting to purify the recombinant candidate receptor protein (cRP) by solubilizing recombinatn Spodoptera frugiperda (Sf9) cells carrying the gene for the histidine-tagged cRP, using 8M urea and purifying the cRP. Additionally, the goal was to achieve direct binding between ChxA and cRP.
Unsuccessful purification attempts: Despite extensive efforts, the purification of cRP could not be achieved using the proposed methods. Solubilization of whole cell proteins with 8M urea followed by nickel affinity chromatography failed to yield purified cRP. The inability to purify cRP suggests challenges related to its large size or other unknown factors.
Failure to establish direct binding: Due to the unsuccessful purification of cRP, direct binding studies between ChxA and cRP could not be conducted. The lack of binding observed in previous attempts also indicates a barrier to establishing this interaction.
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