臺灣博碩士論文加值系統

中文摘要 .............................................................................................................................. i
英文摘要 ............................................................................................................................. ii
目錄 ....................................................................................................................................iii
圖目錄 ............................................................................................................................... vii
表目錄 ............................................................................................................................... xii
第一章 緒論 ............................................................................................................. 1
1.1 心臟衰竭 (Heart Failure) ........................................................................... 1
1.2 前腦利鈉肽、N 端前腦利鈉肽與腦利鈉肽 ............................................. 4
1.3 以胜肽進行辨認 ......................................................................................... 5
1.4 核醣體展示技術 (Ribosome Display) ....................................................... 6
1.5 生物膜干涉技術 (Bio-Layer Interferometry, BLI) ................................... 7
1.6 研究目的 ..................................................................................................... 8
1.7 實驗進程 ..................................................................................................... 8
第二章 實驗方法 ................................................................................................... 10
2.1 轉殖 (Transformation) .............................................................................. 10
2.2 蛋白質純化 ............................................................................................... 10
2.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 ................................................... 12
iv
2.4 布拉德福蛋白質定量法 (Bradford Protein Assay) ................................. 13
2.5 西方墨點法 (Western Blot) ..................................................................... 14
2.6 瓊脂糖凝膠電泳 (Agarose Gel Electrophoresis)..................................... 15
2.7 用於核醣體展示技術之蛋白質製備 ....................................................... 16
2.7.1 UlaG 蛋白質之表現與純化...................................................................... 16
2.7.2 UlaG-proBNP 蛋白質之表現與純化 ....................................................... 17
2.8 核醣體展示技術 ....................................................................................... 19
2.8.1 核醣體展示 DNA 基因庫設計................................................................. 19
2.8.2 UlaG-proBNP 與 UlaG 磁珠製備 ............................................................ 20
2.8.3 核醣體展示技術 (Ribosome Display) 實驗流程 ................................... 21
2.8.4 次世代定序 (Next Generation Sequencing) 與資料處理 ...................... 25
2.9 胜肽合成 ................................................................................................... 25
2.10 重組蛋白 proBNP 之表現與純化 ............................................................ 26
2.11 酵素結合免疫吸附分析法 ....................................................................... 27
2.12 生物膜干涉技術 (Bio-Layer Interferometry, BLI) ................................. 30
2.12.1 原理 ........................................................................................................... 30
2.12.2 擬合模型 ................................................................................................... 30
2.12.3 實驗參數 ................................................................................................... 32
第三章 結果與討論 ............................................................................................... 36
3.1 用於核醣體展示技術之蛋白質製備 ....................................................... 36
3.1.1 UlaG 蛋白質的純化.................................................................................. 36
3.1.2 UlaG-proBNP 蛋白質的純化 ................................................................... 38
3.1.3 以西方墨點法確認 UlaG 與 UlaG-proBNP ............................................ 39
3.1.4 以酵素結合免疫吸附分析法確認 UlaG、UlaG-proBNP ...................... 40
3.1.5 以膠內水解鑑定 UlaG 與 UlaG-proBNP 身份 ....................................... 41
3.2 核醣體展示技術 ....................................................................................... 42
3.2.1 UlaG-proBNP 與 UlaG 磁珠製備 ............................................................ 42
3.2.2 篩選 ........................................................................................................... 44
3.3 次世代定序與胜肽合成 ........................................................................... 47
3.4 重組蛋白 proBNP 之製備 ........................................................................ 50
3.4.1 重組蛋白 proBNP 的表現 ........................................................................ 50
3.4.2 重組蛋白 proBNP 的純化 ........................................................................ 56
3.4.3 以西方墨點法確認重組蛋白 proBNP ..................................................... 62
3.4.4 以酵素結合免疫吸附分析法確認重組蛋白 proBNP ............................. 63
3.4.5 以膠內水解鑑定重組蛋白 proBNP 身份 ................................................ 64
3.5 生物膜干涉技術 ....................................................................................... 65
3.5.1 固定化胜肽對 UlaG 之特性分析............................................................. 65
3.5.2 固定化胜肽對重組蛋白 proBNP 之特性分析 ........................................ 68
vi
3.6 以酵素結合免疫吸附分析法測定胜肽對 proBNP 之親和力 ................ 87
第四章 結論 ........................................................................................................... 90
第五章 未來展望 ................................................................................................... 91
第六章 參考文獻 ................................................................................................... 92
第七章 附錄 ........................................................................................................... 98
7.1 胺基酸序列 ............................................................................................... 98
7.2 FPLC 層析結果 ......................................................................................... 99
7.3 蛋白質身分比對 ..................................................................................... 107
7.4 胜肽 LC-ESI/MS 結果圖 ........................................................................ 108
7.5 實驗藥品與耗材 ..................................................................................... 116
7.6 溶液配置 ................................................................................................. 119
7.7 實驗儀器 ................................................................................................. 128

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