臺灣博碩士論文加值系統

由靈芝 (Ganoderma lucidum) 分離的免疫調節蛋白質LZ-8已證實具有多種免疫調節功能如促進淋巴球增生及抑制全身性過敏反應。本實驗室於2005年以genome walking方式由小孢子靈芝 (G. microsporum) 及拱狀靈芝 (G. fornicatum)釣取到LZ-8相似基因gmi、gfo-1及gfo-2 。之後發現GMI及GFO-1可刺激BALB/c小鼠骨髓樹突細胞分泌IL-12、刺激小鼠巨噬細胞株J774A-1分泌TNF-α以及刺激人類淋巴癌T細胞株Jurkat分泌IL-2。
　　由於前人研究多以細胞株進行免疫調節活性測試，測定的細胞激素種類也較少。故本研究欲以嗜甲醇酵母菌表現LZ-8、GMI、GFO-1及GFO-2，並以小鼠初代細胞探討其免疫調節功能，作為往後進行動物實驗之參考。
　　研究結果顯示，以搖瓶培養並經五倍濃縮，在誘導前菌體細胞濃度可達1.44 ± 0.03 × 1010 cfu/mL (rLZ-8 / H. polymorpha)、2.94 ± 0.23 × 109 cfu/mL (rGMI / P. pastoris) 及8.60 ± 0.14 × 109 cfu/mL (rGFO-1 / H. polymorpha)。以1%甲醇誘導1天後再濃縮純化，可得到23.3 ± 0.1 mg/L rLZ-8、4.3 ± 0.4 mg/L rGMI及8.5 ± 0.2 mg/L rGFO-1。在免疫調節活性部分，rLZ-8、rGMI及rGFO-1均可在未以其他物質刺激的條件下活化免疫系統，亦可刺激小鼠腹腔細胞及脾臟細胞產生細胞激素，具有抑制過敏反應及抑制腫瘤的潛力。與 rLZ-8及rGFO-1相比，rGMI在刺激IL-1β、IL-2、 IFN-γ、IL-4及IL-5的效果有明顯差異，可能與N端的α helix以及前35個胺基酸序列的差異性有關，未來可以點突變探討胺基酸序列的差異對上述細胞激素表現的影響。rGFO-2無法以嗜甲醇酵母菌順利表現，可能因本身基因序列具有高GC比例，密碼子使用率，或是與第60個胺基酸附近的loop序列有關，未來可設計引子修改密碼子，或針對loop的glycine進行點突變。

LZ-8, a fungal immunomodulatory protein (FIP) isolated from Ganoderma lucidum exhibits potent mitogenic effect upon human peripheral blood lymphocytes (PBL) and prevents the production of systemic anaphylaxis reaction in mice. Three lz-8-like genes, gmi, gfo-1 and gfo-2 were successfully isolated from G. microsporum and G. fornicatum previously in our laboratory. GMI and GFO-1 were found to stimulate bone marrow derivated dendritic cells from BALB/c mice, murine macrophage J774A-1 and Jurkat T cells to secret IL-12, TNF-α and IL-2, respectively.
From previous studies, the immune functions of FIPs were estimated based on cell line model, and only a few cytokines were determined. In this study, LZ-8, GMI, GFO-1 and GFO-2 were expressed in methylotrophic yeasts and their immune functions were assayed in BALB/c mice primary cells.
The average production of rLZ-8 and rGFO-1 in H. polymorpha were 23.3 ± 0.1 and 8.5 ± 0.2 mg/L, respectively, while rGMI reached 4.3 ± 0.4 mg/L in P. pastoris. All the rLZ-8, rGMI and rGFO-1 were found to activate the immune system spontaneously and to stimulate BALB/c mice peritoneal cells and splenocytes to secret several cytokines such as IL-1β, TNF-α, IL-6, IL-2, IFN-γ, IL-4 and IL-5. Suggesting that these recombinant proteins exist the potential in suppressing allergic disease and anti-tumor effect. For the stimulation of IL-1β, IL-2, IFN-γ, IL-4 and IL-5, rGMI was significantly different from rLZ-8 and rGFO-1 probably due to the difference in amino acid sequence of N-terminal α helix region. The unsuccessful expression of gfo-2 in methylotrophic yeasts might be explained by the high GC content, different codon usage and the glycine-rich loop.

目錄 I
圖目錄 VI
表目錄 IX
中文摘要 X
Abstract XI
第一章 前言 1
一、 靈芝 1
1. 靈芝概論 1
2. 靈芝藥理活性物質 1
二、 真菌免疫調節蛋白質 3
1. 靈芝免疫調節蛋白質 3
1.1 LZ-8 3
1.2 FIP-gts 4
1.3 GMI 4
1.4 GFO-1 & GFO-2 5
1.5 FIP-gja 6
1.6 免疫調節活性 6
2. 其他真菌免疫調節白質 6
三、 免疫系統簡介 10
1. 先天免疫反應 10
2. 後天免疫反應 11
3. 細胞激素與免疫調節的關係 12
四、 異源表現系統 18
五、 嗜甲醇酵母菌表現系統 21
1. Pichia pastoris 21
2. Pichia methanolica 22
3. Hansenula polymorpha 23
六、 研究動機 26
第二章 材料與方法 28
一、 質體建構 28
1. 插入目標基因 28
2. 置換啟動子 31
3. 修改c-myc epitope 33
二、 轉形 37
1. Escherichia coli DH5α 37
1.1 勝任細胞製備 37
1.2 熱休克轉形 37
2. 嗜甲醇酵母菌 38
2.1 P. pastoris勝任細胞製備 38
2.2 H. polymorpha勝任細胞製備 39
2.3 電穿孔轉形法 39
三、 轉形株篩選 41
1. 抗生素抗性篩選 41
2. 試管小量表現篩選 41
3. 搖瓶表現篩選 42
四、 免疫調節蛋白質的表現與純化 43
1. 蛋白質表現 43
2. 蛋白質濃縮與純化 44
五、 免疫調節蛋白質的分析 46
1. 蛋白質定量 46
2. 蛋白質電泳分析 46
2.1 CBR染色 46
2.2 醣蛋白染色 46
3. GFO-1單株抗體製備 48
3.1 抗原準備及預測 49
3.2 血清效價測試 49
3.3 小鼠免疫 52
3.4 骨髓癌細胞培養 53
3.5 取脾臟、細胞融合 54
3.6 融合瘤細胞株篩選 55
3.7 單株化 56
3.8 誘生腹水 58
3.9 單株抗體純化 59
4. 西方墨點法 60
5. 同源雙體之比例 60
5.1 Native PAGE 60
六、 免疫調節活性測試 61
1. 動物飼養 61
2. 樣品前處理 61
3. 腹腔細胞之取得與培養 61
4. 脾臟細胞之取得與培養 63
5. 細胞激素含量測定 64
6. 統計分析 65
第三章 結果 66
一、 質體建構 66
1. 插入目標基因 66
1.1 pMOXZα-gfo1 66
2. 置換啟動子 69
2.1 pMOXZα-gfo2 69
2.2 pPICZα-gmi-H 71
3. 修改c-myc epitope 73
3.1 pMOXZα-gfo1-H 73
3.2 pMOXZα-gfo2-H 75
3.3 pMOXZα-LZ8-H 77
3.4 pMOXZα-gmi-H 79
二、 轉形株篩選 81
1. pMOXZα-gfo1-H / H. polymorpha 81
2. pMOXZα-LZ8-H / H. polymorpha 81
3. pMOXZα-gmi-H / H. polymorpha 81
4. pMOXZα-gfo2-H/ H. polymorpha 82
5. pPICZα-gmi-H / P. pastoris 82
三、 免疫調節蛋白質分析 88
1. 免疫調節蛋白質的純化與定量 88
1.1 蛋白質純化 88
1.2 蛋白質定量 88
2. 醣蛋白染色 88
3. 西方墨點法 88
4. 單株抗體專一性測試 89
5. 同源雙體之比例 89
四、 免疫調節活性分析 94
1. 免疫調節蛋白質對腹腔細胞分泌細胞激素的影響 94
2. 免疫調節蛋白質對脾臟細胞分泌細胞激素的影響 97
第四章 討論 101
1. 密碼子使用率對免疫調節蛋白質在不同宿主表現的影響 101
2. 免疫調節活性 104
3. 免疫調節蛋白質序列比對 105
第五章 結論 108
第六章 未來展望 109
第七章 參考文獻 110

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