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研究生: 陳彥霖
Chen, Yan-Lin
論文名稱: 建立以單分子螢光顯微技術研究Shugoshin-1蛋白與潛力治療用胜肽間動態交互作用的平台
Development of a single-molecule-fluorescence-microscopy-based platform to study dynamic interactions between Shugoshin-1 and potential therapeutic peptides
指導教授: 李以仁
Lee, I-Ren
口試委員: 楊立威
Yang, Lee-Wei
孫英傑
Sun, Ying-Chieh
口試日期: 2021/08/12
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 62
中文關鍵詞: 胜肽-胜肽交互作用Sgo1PP2A抗微生物胜肽單分子螢光共振能量轉移
英文關鍵詞: Peptide-Peptide interactions, Sgo1, PP2A, Antimicrobial peptides, smFRET
DOI URL: http://doi.org/10.6345/NTNU202101171
論文種類: 學術論文
相關次數: 點閱:104下載:6
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  • 蛋白質-蛋白質交互作用(Protein-Protein interactions, PPIs)促進大部分生物機能,蛋白質區域與區域之間的交互作用(Domain-Domain interactions, DDIs)引導功用性蛋白在特定區域作用,交互作用必須通過蛋白質的二級結構進行,二級結構中螺旋結構穩定且單純,探討螺旋結構之間的交互作用能夠得到更精確的結果。
    Shugoshin 1 (Sgo1)和蛋白磷酸酶2A (PP2A)之間的結合研究說明Sgo1募集PP2A在保護姊妹染色體中有重要的作用。為了發展一系列抗微生物胜肽,我們擷取Sgo1-PP2A交互作用的片段,將目標Sgo1胜肽和DNA交聯,並經由與生物素標記的互補DNA結合而固定在單分子檢驗平台上,並在緩衝溶液中添加螢光標記的游離潛力治療用胜肽進行交互作用,利用單分子螢光顯微鏡研究胜肽-胜肽交互作用動力學。和傳統的方法比較,需要的胜肽濃度相對較低(< 100 nM),使我們可以對潛力治療用胜肽進行更廣泛的篩選。

    Protein-Protein Interactions (PPIs) facilitate many biological functions, and Domain-Domain Interactions (DDIs) induce functional protein activity in specific regions. The interactions between domains are usually dictated by their secondary structures . Helical structure is relatively simple and stable , hence , becomes a suitable candidate for studying domain-domain interactions . Previous study of Shugoshin 1(Sgo1)-Protein Phosphatase 2A(PP2A) binding demonstrated that recruitment of PP2A by Sgo1 plays an important role in the protection of sister chromatid , Hence , becomes a potent target for the development of antimicrobial peptides. We extract the fragments of Sgo1-PP2A interaction and establish a single-molecule-fluorescence-microscopy-based platform for studying the peptide-peptide interaction dynamics. Target peptide (Sgo1)-DNA hybrid was used in the immobilized single-molecule assays and interacted with the fluorescence-labeled free potential therapeutic peptides in buffer solution. The required concentration (< 100 nM) of peptides is relatively low compared to conventional methods, allowing us to perform a wider range of screening on the potential therapeutic peptides.

    致謝 i 摘要 ii Abstract iii 目錄 iv 圖目錄 vi 表目錄 vii 第一章 緒論 1 1.1 前言 1 1.2 蛋白質-蛋白質交互作用(Protein-Protein Interactions, PPIs) 2 1.3 區域-區域交互作用(Domain-Domain Interactions, DDIs) 3 1.4 治療用胜肽數據庫(Therapeutic Peptide-DataBank, TP-DB) 7 1.5 抗微生物胜肽(Antimicrobial Peptides, AMPs) 8 1.6 肝細胞癌(Hepatocellular carcinoma, HCC) 10 1.7 研究動機 11 第二章 實驗方法與儀器 13 2.1胜肽和DNA接合與螢光標記PP2A胜肽 13 2.1.1胜肽-DNA接合物製備 13 2.1.2 PP2A胜肽標記 14 2.2實驗儀器與原理 16 2.2.1逆相高效液相層析(Reverd Phase High-Performance Liquid Chromatography, RP-HPLC) 16 2.2.2液相層析質譜儀(Liquid Chromatography-Mass Spectrometry, LC-MS) 17 2.2.3基質協助雷射誘導結晶/離子化-飛行時間質譜儀(Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry, MALDI-TOF-MS) 18 2.2.4單分子螢光共振能量轉移 19 2.2.5全內反射螢光顯微鏡 23 2.3單分子實驗器材與實驗樣品製備流程 25 2.3.1實驗用玻片槽處理與組裝 25 2.3.2顯像緩衝溶液(Image Buffer) 26 2.3.3實驗方法與流程 28 2.4數據處理與分析 32 第三章 實驗結果與討論 34 3.1實驗設計 34 3.2 Sgo1胜肽DNA連接物產物鑑定 35 3.2.1 HPLC純化Sgo1胜肽DNA連接物 35 3.2.2 MALDI-TOF MS鑑定 35 3.2.3 LC-MS鑑定 36 3.2.4目標Sgo1胜肽與DNA共定位 39 3.3目標Sgo1胜肽與PP2A胜肽特定的結合 41 3.3.1透析純化標記上螢光分子的PP2A胜肽 41 3.3.2透析法純化PP2A胜肽與目標Sgo1胜肽結合 42 3.3.4溫度對胜肽結構的影響 49 3.3.5 PP2A胜肽與Sgo1胜肽之間特定的結合 50 3.4 PP2A-Sgo1結合之動力學 51 3.5 Sgo1-PP2A交互作用模型 55 第四章 結論與未來展望 57 參考文獻 59

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