摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VI
壹、 前言 1
一、 研究背景 1
二、 研究目的 1
貳、 文獻回顧 2
一、 反轉錄酶 (reverse transcriptase) 2
二、 大腸桿菌之蛋白質表現系統 2
1. 大腸桿菌表現載體 2
2. E. coli BL21 (DE3) 3
3. E. coli BL21 StarTM (DE3) 3
4. ClearColiTM BL21 (DE3) 3
三、 分子伴護蛋白 4
四、 發酵技術 4
1. 發酵槽種類 4
2. 發酵槽培養方式 5
2.1. 連續式發酵 (continuous fermentation) 5
2.2. 批式發酵 (batch fermentation) 5
2.3. 饋料批式發酵 (fed-batch fermentation) 5
五、 蛋白質濃縮 6
1. 透析膜濃縮法 6
2. 冷凍乾燥濃縮法 6
3. 超濾膜濃縮法 6
4. 其他濃縮法 6
六、 酵素穩定性與活性保存 7
1. 海藻糖 7
2. 金屬離子 7
參、 實驗設計與流程 8
肆、 材料與方法 9
一、 實驗材料 9
1. 菌株 9
2. 載體 9
3. 培養基 9
4. 抗生素 9
5. 標準品 9
6. 市售套組 9
7. 化學藥品 10
二、 實驗設備 11
三、 實驗方法 13
1. 製備電穿孔之勝任細胞 13
2. 電穿孔轉形作用 13
3. 菌種保存 13
4. 抽取質體 DNA 13
5. 細胞破碎 14
6. SDS-PAGE 15
7. 目標重組 RT 在大腸桿菌的表現 16
8. 發酵槽生產重組 RT 18
9. 目標重組 RT 的萃取與純化 19
10. 重組 RT 的蛋白質定量 21
11. 重組 RT 的酵素活性測定 22
12. 重組 RT 的儲存安定性試驗 24
伍、 結果與討論 25
一、 重組蛋白之製備最適條件探討 25
1. 最佳表現菌株之篩選 25
2. 最適IPTG誘導濃度之篩選 25
3. 最適chaperone之篩選 25
二、 利用高密度細胞培養添加饋料之方式生產重組RT 26
三、 重組RT之親和性管柱純化及儲存安定性試驗 27
陸、 結論 29
柒、 參考文獻 30
捌、 圖表 34
玖、 附錄 55

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