臺灣博碩士論文加值系統

摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
壹、 前言 1
(一) 研究背景 1
(二) 研究目的 1
貳、 文獻回顧 3
(一) 分子量標準蛋白 3
1. 重組分子量標準蛋白 3
2. 預染分子量標準蛋白 3
(二) 基因重組蛋白的表現 3
1. 表現系統 3
2. 大腸桿菌宿主菌株 4
3. 表現載體 5
4. 轉殖技術 6
5. 質體的穩定度 6
(三) 重組融合蛋白 7
1. 簡介 7
2. 連接肽 7
3. PIPE cloning 7
(四) 發酵槽饋料批式發酵 8
1. 生物反應器 8
2. 高細胞密度培養 8
3. 發酵槽之運作模式 9
4. 饋料成分 9
(五) 蛋白質之純化 10
1. 蛋白質萃取 10
2. 硫酸銨分劃 10
3. 親和性層析 11
4. 離子交換層析 11
5. 膠體過濾層析 11
(六) 電泳中的蛋白質染色 12
1. 傳統染劑 12
2. 螢光染劑 12
3. 反應性染劑 12
參、 實驗架構 13
肆、 材料與方法 15
(一) 實驗材料 15
1. 菌株 15
2. 質體 15
3. 市售套組 15
4. 藥品 15
(二) 實驗器材 17
(三) 培養基配製 18
1. Terrific broth (TB) 培養基 18
2. Lysogeny broth (LB) 平板培養基 18
3. SOC培養基 19
(四) 實驗方法 19
1. 抽取質體DNA 19
2. DNA電泳 19
3. PCR擴增基因片段及載體 20
4. 限制酶剪切 20
5. 製備電穿孔之大腸桿菌勝任細胞 20
6. 電穿孔轉形作用 21
7. Colony PCR 21
8. 菌種保存 21
9. 搖瓶之細菌培養 21
10. 發酵槽之細菌培養 22
11. 細胞破碎 22
12. SDS-PAGE 23
13. 鎳離子螯合親和性層析 24
14. 陰離子交換層析 25
15. 膠體過濾層析 25
16. 蛋白質定量 26
17. 蛋白質染色 26
18. 生產成本計算 27
伍、 結果與討論 29
(一) 重組目標蛋白之表現 29
(二) 重組質體之建構 29
1. Tre-PG融合基因 29
2. MBP基因 30
(三) Tre-PG重組蛋白之製備最適條件探討 30
1. 最佳表現菌株 30
2. 最適IPTG誘導濃度 31
3. 最適誘導溫度及時間 31
4. 菌體破壁 31
5. 鎳離子螯合親和性管柱純化 32
6. 陰離子交換管柱純化 32
7. 膠體過濾管柱純化 33
(四) His-Tre-PG之製備 33
1. 質體製備 33
2. 蛋白質純化 33
(五) 以高密度細胞培養添加批式饋料之方式生產重組蛋白質 34
(六) 重組蛋白質染色條件之最適化探討 35
1. 蛋白質還原 35
2. 最適染劑濃度 36
3. 最適染色時間 36
4. 最適染色緩衝液組成 36
5. 最適染色緩衝液pH值 36
6. 最適染色溫度 37
(七) 彩色預染分子量標準蛋白試劑之製備 37
1. 試劑組成 37
2. 生產成本 37
陸、 結論 39
柒、 參考文獻 41
捌、 圖表 49

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