臺灣博碩士論文加值系統

腫瘤細胞是正常細胞基因突變所產生，而這些基因發生改變的細胞常會產生異常的表面抗原，然而免疫系統卻無法有效的對這些異常的抗原產生反應，亦即腫瘤細胞產生了免疫耐受性(tolerance)，在癌症的免疫治療中，必需克服腫瘤細胞產生的免疫耐受性才能有效的殺死癌細胞。人類&;#21554;&;#21722;胺2,3-雙加氧&;#37238;(Indoleamine 2,3-dioxygenase；IDO)是色胺酸L-Tryptophan (L-Trp)代謝路徑的速率決定酵素，近來也被報導是樹突細胞(Dendritic cells；DCs)的免疫負向調控因子。當樹突細胞表現IDO時，會抑制T細胞的增生與活化，而免疫調節T細胞(T Regulatory cells;Treg)則被誘發增殖，進而抑制免疫反應。IDO會在腫瘤細胞內和腫瘤浸潤淋巴結創造一個免疫耐受性環境，並在這二者中抑制T細胞和強化局部Treg的免疫抑制功能。因此我們假定抑制IDO活性可能增強腫瘤免疫治療的有效性。我們利用E. coli系統表現IDO重組蛋白並以快速液態層析儀(Fast protein liquid chromatography, FPLC)系統進行純化以作為免疫抗原對BALB/c小鼠進行皮下或脾臟免疫，在誘發出對IDO蛋白具專一性的抗體後，取脾臟細胞與骨髓瘤細胞(NS-1)進行細胞融合，接著以Soft agar技術選殖出可分泌IDO抗体的融合瘤細胞株。藉由ELISA和西方墨點實驗的驗證，共篩選出3株對IDO具有專一性的單株抗體。最後進行Domain mapping以確定抗体在IDO抗原上的辨識區域。

The enzyme indoleamine 2,3-dioxygenase (IDO) attracted scientist special attention by it’s ability to contribute tumors escape from the host’s immune response against them. IDO starves T cells from tryptophan by this important amino acid catabolizing to N-formyl-kynurenine, which makes these T cells unable to mounting appropriate immune responses against tumors. IDO-dependent T cell suppression by dendritic cells due to tryptophan catabolism have effects on T cell proliferation, differentiation, effector functions, and viability. IDO helps suppression of T cells and enhancement of local Treg-mediated immunosuppression by create a tolerogenic milieu in the tumor and the tumor-draining lymph nodes directly. Therefore, strategies to block IDO activities might enhance the effectiveness of tumor immunotherapy.
In this study, we refer to the NCBI database to construct and express various truncated IDO proteins flanks with 6-histidine tag. We also obtain 5 specific hybridoma clones screening via ELISA assay. Three of them response to IDO by Western blot indicate these specific recognize the linear epitope. Others response to IDO by dot blot but not western blot imply those recognize stereo-structure of IDO protein.

中文摘要 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .i
英文摘要 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ii
目錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii
表目錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vii
圖目錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vii
附錄目錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viii
縮寫檢索表 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix
第一章 緒論 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
1-1 Indoleamine 2,3-dioxygenase (IDO) 之生物功能簡介 . . . . . . . . . . . .1
1-2 IDO與適應性免疫調節機制 . . . . . . . . . . . . . . . . . . 2
1-2-1 體內免疫系統對抗外來抗原的方式 . . . . . . . . . . . . . . . . . . . . . . .2
1-2-2 IDO的免疫負向調節功能 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1-2-3 IDO抑制免疫之分子調控機制 . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1-3 腫瘤的免疫逃脫與免疫治療 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1-4 研究動機與目的 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
第二章 材料與方法 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2-1 IDO抗原的製備 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2-1-1 IDO 基因之取得 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2-1-2 表現載體構築 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2-1-2-1 引子(Primer)設計 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2-1-2-2 插入DNA(Insert DNA)聚合酶連鎖反應( Polymerase Chain
Reaction；PCR ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2-1-2-3 PCR產物(insert DNA)的純化 . . . . . . . . . . . . . . . . . . . . . . . 10
2-1-2-4 限制酶處理( Restriction enzyme digestion ) . . . . . . . . . . . . .11
2-1-2-5 接合反應 ( Ligation reaction ) . . . . . . . . . . . . . . . . . . . . . . . 12
2-1-2-6 轉形作用(Transformation) . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2-1-3 Colony PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2-1-4 小量質體製備 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2-1-5 定序 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2-1-6 利用BL21-CodonPlus表現IDO蛋白 . . . . . . . . . . . . . . . . . . . . 16
2-1-7 蛋白純化 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2-1-7-1 純化用buffer之製備 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2-1-7-2 純化流程 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2-2 製備IDO單株抗體 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2-2-1 小鼠免疫 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2-2-2 小鼠脾臟淋巴細胞之回收 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2-2-3 細胞融合 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2-2-3-1 細胞培養液 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2-2-3-2 細胞融合流程 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2-2-4 以半固體瓊脂培養基(soft agar)進行單株化 . . . . . . . . . . . . . . .20
2-2-5 酵素連接免疫吸附法(ELISA assay) . . . . . . . . . . . . . . . . . . . . . . 21
2-2-6 融合瘤細胞的擴大培養及抗體分類分析 ( Isotyping ) . . . . . . .23
2-2-7 抗體生產 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2-2-8 利用protein A 管柱純化單株抗體 . . . . . . . . . . . . . . . . . . . . . . . 24
2-2-8-1 配製Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2-2-8-2 抗體純化流程 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2-3 抗原決定區辨識 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2-3-1 構築人類截短型(truncate) IDO重組蛋白並小量表現 . . . . . . 24
2-3-2 以SDS-PAGE確認分子量 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2-3-2-1 配製Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2-3-2-2 以SDS-PAGE電泳分析各截切片段蛋白分子量 . . . . . . . 27
2-3-3 以Western 進行單株抗體 Mapping . . . . . . . . . . . . . . . . . . . . . . 28
2-3-3-1 Buffer配置 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2-3-3-2 抗原決定區辨識 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2-3-4 圓漬點墨法 (Dot blot) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

第三章 結果與討論 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3-1 表現載體的構築 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3-1-1 以SKOV3 cDNA選殖出IDO全長基因 . . . . . . . . . . . . . . . . . . 31
3-1-2 構築全長與截短型IDO於pET-21b表現載體 . . . . . . . . . . . . . 31
3-2 IDO蛋白的表現及純化 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3-2-1 E. coli表現寄主的選用 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3-2-2 免疫用IDO蛋白表現及純化 . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3-2-3 截短型IDO蛋白表現 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3-3 抗體的產生 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3-3-1 免疫後血清抗體力價測試 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3-3-2 以半固體瓊脂培養基(soft agar)挑選單株細胞 . . . . . . . . . . . . . 34
3-3-3 以ELISA鑑定IDO單株抗體 . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3-3-4 使用SCID小鼠生產抗體 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3-4 以Western進行單株抗體免疫分析(Domain mapping) . . . . . . . . . . . 36
第四章 結論與展望 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
參考文獻 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
表 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
圖 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
附錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

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