目錄
摘要 I
Abstract II
致謝 III
圖片目錄 IV

第一章 慢性發炎簡介：以類風濕性關節炎為例 1
1-1. 類風濕性關節炎簡介 1
1-2. 類風濕性關節炎致病源由 2
1-3. 類風濕性關節炎疾病進程 2
1-4. 自體免疫T細胞在類風濕性關節炎扮演的角色 4
1-5. 發炎型酵素在類風濕性關節炎扮演的角色 5
1-6. 現行藥物- Drug A 6
1-7. Drug A減緩發炎性疾病機制 6
1-8. 實驗設計與驗證 9
第二章 研究方法及使用材料 11
2-1. 材料-常用化學藥品 11
2-2. 材料-限制酵素 11
2-3. 材料-抗體 11
2-4. 建構SCT004以及SCT005重組蛋白之載體 12
2-4-1. 從脾臟細胞萃取RNA 12
2-4-2. RNA分離步驟 13
2-4-3. 將RNA反轉錄成cDNA 13
2-4-4. 藉由PCR放大所需的嵌入基因 14
2-4-5. 限制酵素剪切以及確認 14
2-4-6. 膠體純化剪切後的載體以及嵌入基因 15
2-4-7. 製作勝任細胞 16
2-4-8. 將嵌入基因接入載體 16
2-4-9. 轉移載體至勝任細胞 16
2-4-10. 管柱層析純化載體 17
2-4-11. 限制酵素剪切 pLNCX-Fc-IgG1以及確認 17
2-4-12. 限制酵素剪切pLNCX- Fc-IgG1-substrate sequence II-Drug A”-His 18
2-4-13. 限制酵素剪切pLNCX-Protein TM-substrate sequence II-Drug A”-His 18
2-4-14. 經由酒精沈澱取得無菌的表現載體 20
2-5. 表達並且生產純化SCT004以及SCT005重組蛋白 21
2-5-1. 將表現載體轉染至真核細胞 21
2-5-2. 預先準備鎳離子親和性樹脂 21
2-5-3. 以鎳離子親和性樹脂純化重組蛋白 21
2-5-4. 蛋白質電泳 22
2-5-5. Coomassie blue染色 22
2-5-6. Bicinchoninic acid蛋白質定量(BCA assay ) 23
2-5-7. 西方墨點法 23
2-5-8. 點漬法 23
2-6. 確認SCT004以及SCT005重組蛋白的特性 24
2-6-1. 使用特定催化酵素活化重組蛋白 24
2-6-2. HEK293 Ligand B細胞準備 24
2-6-3. 競爭型流式細胞儀分析 25
2-6-4. 直接型流式細胞儀分析 25
2-6-5. 細胞依附型酵素連結免疫吸附分析(Cell-Based ELISA) 26
第三章 結果 28
3-1. 設計SCT004以及SCT005重組蛋白 28
3-2. 生產與純化SCT004以及SCT005重組蛋白 29
3-3. SCT004以及SCT005重組蛋白是否能被剪切移除遮蔽區域 31
3-4. 初步測試新型重組蛋白活化前後的結合能力 33
3-5. 以cell-based ELISA計算重組蛋白活化前後的活性差異 35
第四章 結果與討論 41
4-1. SCT004與SCT005設計與優勢 41
4-2. SCT004與SCT005是否可在病患患部中活化 42
4-3. SCT004與SCT005 可能的缺點 43
Reference: 45
實驗使用溶液 47
英文簡寫對照表 49

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