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研究生: 陳姿婷
Tz-Ting Chen
論文名稱: 設計與合成一系列的胜肽以抑制Galectin-3蛋白形成液滴狀態
Design and Synthesis of Different Peptides Against Galectin-3 Condensate Formation
指導教授: 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
口試委員: 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
黃介嶸
Jie-Rong Huang
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 84
中文關鍵詞: 半乳糖凝集素3醣結合蛋白胜肽液滴溶酶體
外文關鍵詞: Galectin-3, Galactose-binding protein, Peptide, Condensate, Lysosome
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  • 摘要 iii Abstract iv 致謝 v 目錄 i 圖目錄 iv 表目錄 vi 附圖目錄 vii 縮寫表 viii 第一章 緒論 1 1.1 生物體中的液-液相分離(LLPS) 1 1.2 Galectin-3之結構及聚集機制 3 1.3 Galectin-3在小膠質細胞中的功能與神經退化性疾病之關聯 5 1.4 研究動機及目的流程 6 第二章 實驗儀器與材料 7 2.1 實驗儀器 7 表2-1 實驗儀器表 7 2.2 實驗材料 9 表2-2 實驗材料表 9 第三章 實驗方法 12 3.1 胜肽樣品製備 (合成、純化與鑑定) 12 3.1.1固相胜肽合成 (Solid-Phase Peptide Synthesis, SPPS) 12 3.1.2 胜肽染劑偶合 (Peptide-Dye coupling) 14 3.1.3高效液相層析(High-Performance Liquid Chromatography, HPLC) 15 3.1.4 基質輔助雷射脫附游離質譜法 (MALDI Mass Spectrometry) 16 3.2 重組蛋白表達、純化與鑑定 17 3.2.1 Galectin-3表達 17 3.2.2 Sumo Protease表達及純化 19 3.2.3 FUS 蛋白表達、純化與鑑定 21 3.2.4 SDS-PAGE及西方墨點法 22 表3-1 Western blot試劑與配方表 23 3.3 液滴性質分析 25 3.3.1 混濁度測試 (Turbidity assay) 25 3.3.2 微分干涉相差顯微鏡 (Differential interference contrast, DIC) 26 3.4 細胞培養與繼代 28 3.5 免疫螢光染色 29 3.6 光漂白螢光恢復術 31 3.6 核磁共振(NMR) 32 第四章 實驗結果與討論 33 4.1 Gal-3胜肽材料的設計 33 表4-1胜肽序列與名稱表 35 4.2 胜肽的合成、純化與鑑定 35 表4-2 胜肽名稱與理論/實際測得分子量 35 4.3 藉由脂多醣建立Gal-3體外液滴模型討論液滴與Gal-3結合胜肽相互作用 36 4.3.1利用西方墨點法、混濁度、液滴模型鑑定Gal-3重組蛋白 36 表4-3 純化Gal-3收集樣品組成表 37 4.3.2 利用Gal-3與脂多醣(Lipopolysaccharide)之相互作用建立體外Gal-3蛋白液滴模型 38 4.3.3 胜肽液-液相分離之性質鑑定 40 表4-4 胜肽染劑表 40 4.4 建立與鑑定體外FUS凝集物模型與胜肽相互作用探討Gal-3結合胜肽對於液滴抑制專一性 46 4.4.1利用SDS-膠體電泳及西方墨點法鑑定FUS重組蛋白 46 4.4.2 利用FUS凝集物模型與胜肽相互作用探討Gal-3結合胜肽對於液滴狀態抑制專一性 47 4.5 建立與鑑定細胞內蛋白液滴模型及胜肽探針與蛋白質之相互作用 48 4.5.1建立LLOMe觸發Gal-3細胞液滴模型 48 4.5.2利用光漂白螢光恢復術鑑定溶酶體破壞劑對於SM826小膠質細胞內穩定表達Gal-3-eGFP蛋白液滴性質 49 4.5.3利用螢光胜肽探針在細胞內Gal-3之相互作用探討胜肽對於液-液相分離的抑制能力 51 第五章 總結 55 第六章 未來展望 56 本篇論文 56 附錄 57 附表1 利用噬菌體展示技術胜肽 57 參考資料 66  

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