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研究生:吳函蒼
研究生(外文):Han-Tsang Wu
論文名稱:製備同位素標定之ApolipoproteinE以供NMR分析
論文名稱(外文):Preparation of isotope-labeled Apolipoprotein E for NMR analysis
指導教授:黃憲斌
指導教授(外文):Hsien-bin Huang
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:93
語文別:中文
論文頁數:56
中文關鍵詞:NMRApolipoprotein E
外文關鍵詞:Apolipoprotein ENMR
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Apolipoprotein (ApoE) 被認為與阿茲海默症的主要病狀:神經突斑塊和神經纖維纏結的發生有關係,在人體中ApoE主要有三種對偶基因:E2、E3和E4這三種不同的isoforms具有不同的N-terminal domain以及相同的C-terminal domain,而N-terminal domain決定不同isoform的弁遄AApoE4被認為是造成晚發型阿茲海默症的危險因子,相反地,ApoE2與ApoE3則具有能抑制或延緩神經突斑塊的形成,然而,ApoE N-terminal domain的結構已知為4個?helix以反平行的方式組成束狀,但是其C-terminal domain之結構至今仍未知道,所以我們的研究目的為解出ApoE C-terminal domain之結構以及瞭解ApoE3與ApoE4在結構上之差異。我們首先純化出高純度同位素標定之ApoE C-terminal domain以供NMR分析它的立體空間結構,從結果可得知確實能在NMR分析下獲得正確的chemical shift,另外我們同時純化未標定之ApoE3和ApoE4 N-terminal domain期望能藉由NMR去探討不同的ApoE N-terminal domain 與其C-terminal domain之間的鍵結,目前protein已經純化完畢,並且將在進行NMR的研究。
Apolipoprotein E (ApoE) has been implicated in the formation of the extracellular neuritic plaques and the intracellular neurofibrillary tangles, which are highly associated with pathological severity of Alzheimer’s disease. In human, there are three APOE alleles, E2, E3 and E4, and the differences between isoforms are located at their amino terminal domains, which also determine the function of each ApoE isoform. ApoE4 is one of the risk factors determining the developing late-onset family of Alzheimer’s disease. In contrast, ApoE2 and ApoE3 could arrest extracellular neuritic plaques formation. The structure of ApoE N-terminal domain is consisted of a four-helix bundle with antiparallel arrangement, but the structure of the C-terminal domain remains unknown. The purpose of our studies is to analyze the structure of ApoE C-terminal domain and to determine the structural difference between ApoE3 and ApoE4. We obtained highly purified isotope-labeled ApoE C-terminal domain for NMR analysis. In addition, we also prepared the N-terminal domains of ApoE3 and ApoE4 for determination of the structural differences between ApoE isoforms by NMR analysis. At present, we had accomplished all the protein preparation and the NMR analysis is underway now.
中文摘要……………………………………………………………………….i
英文摘要………………………………………………………………………ii
目錄………………………………………………………………….iii
圖目錄………………………………………………………………………vi
表目錄…………………………………………………………….…vii
第一章 緒論………………………………………………………………….....1
第二章 實驗材料與方法…………………………………………………...…6
2-1 實驗材料………………………………………………………………..….6
2-1.1 化學藥品…………………………………………………………...…...6
2-1.2 M9培養液配方…………………………………...............................….6
2-1.3緩衝溶液之配置…………………………………………………..........8
2-1.4 Ni2+-column的製備……………………………………………….……9
2-1.5 C-18 column的平衡……………………………………………….…...9
2-2 實驗方法…………………………………………………………..……..10
2-2.1 質體pET32a-ApoE 195-299之構築………………………………...10
2-2.2 質體pET32a-ApoE 之構築………………………………………....10 2-2.3 DNA片段的回收或純化……………………………………….…..11
2-2.4 勝任細胞 (Competent Cell) 之制備…………………………....…...12
2-2.5 轉形 (transformation) 作用………………………………….….…...12
2-2.6 質體DNA之抽取………………………………………….…………12
2-2.7藉由定點突變 ( site-directed mutagenesis) 的方式構築pET32a- ApoE3N與pET32a-ApoE4N……………………………………..….……..13
2-2.8表達重組蛋白…………………………………………………......…...14
2-2.8a表達未標定之重組蛋白…………………………………………....14
2-2.8b表達具同位素15N、13C和2H標定之ApoE195-299 融合蛋…...15
2-2.8c表達具同位素15N和13C標定ApoE3N與ApoE4N融合蛋白.....15
2-2.9 透析及冷凍乾燥……………………………………………….......….16
2-2.10 重組蛋白的純化步驟………………………………………..…..…..16
2-2.10a 藉由Ni2+-column純化具有His-Tag 之重組蛋白…………...…16
2-2.10b 藉由thrombin的切割以切除融合蛋白…………………………17
2-2.10c 藉由Ni2+-column來純化不具有Tag之重組蛋白…….…….….17
2-2.10d 藉由HPLC來純化蛋白…………………………………….…...17
第三章 實驗結果.............................................................................................19
3-1 同位素15N、13C和2H標定ApoE195-299之製備……………………19
3-1.1 表達具同位素15N、13C和2H標定ApoE195-299 融合蛋白….…19
3-1.2純化具同位素15N、13C和2H標定ApoE195-299…………………19
3-1.3藉由NMR 分ApoE195-299之立體結構…………………………..21
3-2 ApoE3N與ApoE4N之製備…………………………………………….21
3-2.1 表達ApoE3N以及ApoE4N 融合蛋白.............................................21
3-2.2純化ApoE3N以及ApoE4N………………………………….……...22
3-2.3 以NMR分析15N和13C標定的ApoE3N之結果………………….23
3-3 D2O之再生與利用……………………………………………………….23
第四章 討論………………………………………………………………….24
第五章 參考文獻…………………………………………………………….27
附圖…………………………………………………………………………...31
附表…………………………………………………………………………...47
附錄…………………………………………………………………………...48
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