臺灣博碩士論文加值系統

雖然導致阿茲海默症 ( Alzheimer's Disease, AD ) 的病因還未能完全了解，但目前的研究認為它主要是由於類澱粉胜肽 ( Amyloid β- protein, Aβ) 於腦中沉澱形成老人斑 ( Senile plaque, SP ) 而造成細胞死亡。類澱粉胜肽的沉澱主要是由39至42的胺基酸殘基構成 ( Aβ39-42 )。老人斑內的Aβ是 β-Amyloid precursor protein ( APP ) 經?, β- 和γ-secretases 切割的產物。過去研究指出 Aβ42 被切下來後其結構會由溶解度較高的 random coil 結構轉變成β-sheet 結構，之後便聚集沉澱在神經細胞外，但是對於其沉澱的過程所知甚少。利用 NMR 研究 Aβ 的結構有利於去發展出與 Aβ 胜肽結合之胜肽或化合物以期能有效解決斑塊形成的問題。但是Aβ 胜肽價格高。本實驗室已經成它a純化 Aβ 胜肽，但是所得到的產率很低。我的研究目的是改良純化方法並且增加產率。

Although the etiology of Alzheimer’s disease ( AD ) is not completely realized, recent research suggests that amyloid deposits is central to the disease process. Amyloid deposits which mainly comprise aggregation of a 39-42 residue peptide called β-amyloid peptide (Αβ) form neuritic plaques in extracellular neuron cell. Aβ is derived from the larger amyloid precursor protein (APP), upon cleavages byβ- andγ-secretases. Recent studies have revealed that the conformational change of Αβ peptide from an random coil structure to an β-sheet, in turn leading to aggregation and fibrillar formation. But the process of aggregation remained unclear. Using NMR to studyΑβ structure has an advantage of developing a tool for testing peptides or chemicals whether they can prevent the formation of aggregation. However, the commercialΑβis expensive. My lab has successfully prepared theΑβbut the yield is too low. My goal is to improve the purification methods and increase the yield forΑβpurification.

目錄

英文摘要……………………………………………………………8
中文摘要……………………………………………………………9
第一章 緒論………………………………………………………10
1-1 背景簡介………………………………………………….10
1-2 文獻探討………………………………………………….11
1-3 實驗動機………………………………………………….15
第二章 實驗材料與方法…………………………………………16
2-1 實驗材料與藥品………………………………………….16
2-2 實驗方法………………………………………………….16
2-2.1 蛋白質的表達………………………………………..16
2-2.2 Ni+2 –column的製備…………………………….......17
2-2.3 野生型Αβ融合蛋白質的純化………………….......17
2-2.4 Αβ的純化……………………………………….......18
第三章 實驗結果…………………………………………………..20
3-1 野生型Αβ融合蛋白質的表達和純化……………….......20
3-2 透析緩衝液的pH值和野生型Aβ的沉澱
速率之間關係…………………………………………......20
3-3 15N-標定Αβ融合蛋白質的表達和純化…………………21
3-4 15N- 標定Αβ的純化……………………………………21
3-5 經 Factor Xa作用後之Αβ融合蛋白質
的SDS-PAGE 分析…………………………………....22
3-6 Αβ的逆相高效能液相層析儀
( Reverse-phase HPLC ) 純化後的結果……………...23

第四章 討論………………………………………………………24
參考文獻…………………………………………………………..25
附錄一 緩衝溶液系統……………………………………………42
附錄二 蛋白質表達溶液系統……………………………………43
附錄三 其他試劑…………………………………………………45









附圖目錄

圖 一. Aβ融合蛋白質於LB培養基中之小量表達結果…...31
圖 二. Aβ融合蛋白質於LB培養基中之大量表達結果…...32
圖 三. 透析緩衝液 pH和沉澱速率之關係………………….33
圖 四. 野生型Aβ經逆相高效能液相層析儀………………..34
圖 五. 15N-標定Aβ融合蛋白質於M9培養基中
之大量表達結果………………………………………35
圖 六. 利用Factor Xa切割Aβ融合蛋白質的情形………..36
圖 七. 15N-標定Aβ經逆相高效能液相層析儀
( Reverse-phase HPLC) 純化後的結果…………….37


表一. 以1公升之培養基表達經純化後的Aβ融合蛋白質、 胜肽 的產量表…………………………………………………………..38
附圖 一 APP 簡圖……………………………………………….39
附圖 二 Aβ fibrillogenesis 模式示意圖………………………40
附圖 三 Aβ表達質體示意圖……………………………………41

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