臺灣博碩士論文加值系統

靈芝為中國著名的藥用真菌，含有許多對人體有益的成分，如多醣體、三萜類及真菌免疫調節蛋白質 (Fungal Immunomodulatory Proteins, FIPs)。本實驗室自小孢子靈芝 (Ganoderma microsporum) 選殖出真菌免疫調節蛋白質基因GMI，並以嗜甲醇酵母菌Pichia pastoris表達系統生產。GMI已證實具刺激人類T細胞株分泌IL-2、降低發炎因子分泌、抑制腫瘤轉移及誘發腫瘤細胞凋亡等效果。GMI雖具多種功效，卻可能因彼此的交互作用，難以用於藥物發展。因此探討GMI與細胞表面受體結合的機制及與蛋白質結合相關的胺基酸是極為重要的。過去研究發現FIPs會使山羊血球凝集，生理活性與已知的凝集素 (Lectins) 相似，兩者皆為分子量小卻能對不同細胞造成不同作用的蛋白質。本研究擬探討GMI作用機制是否與凝集素相似，同樣藉由與細胞表面受體的醣分子結合，而影響其下游生理活性。目前結果顯示加特定醣分子，GMI活化Jurkat細胞分泌IL-2的功能會受影響，但抑制肺癌細胞A549生長的功能卻不受影響；以澱粉酶競爭細胞表面醣分子，可得到相似的結果。由此推測GMI是藉由與醣分子的結合而產生免疫活化的功能。此外，芳香族的胺基酸在蛋白質與醣結合能力扮演著重要的角色，其中又以色胺酸最為重要。因此將GMI上三個色胺酸置換成丙胺酸或甘胺酸。其中唯有將三個色胺酸同時至換掉時才會影響抗癌功能，但GMI-W25G、GMI-W110A、GMI-W12A/W25G/W110A會影響免疫活化功效。總而言之，在本研究中發現GMI活化Jurkat細胞分泌IL-2的功能與醣分子結合有關，而其序列上第25及110個胺基酸似乎扮演關鍵的角色。

Ganoderma is a traditional Asian medicine, which contains many bioactive compounds, such as triterpenoids, polysaccharides, and fungal immunomodulatory proteins (FIPs). GMI is a FIPs found in Ganoderma microsporum, and successfully produced by pichia pastoris, a heterologous protein expression system. It was proved that recombine GMI was able to stimulate Jurkat T cell secreting IL-2, reduced inflammation, and killed cancer cell. GMI was multifunction, but it is difficult to become a drug because of the interactions of those functions. As a result, the detail mechanism of how GMI interacts with the cell surface receptors and which amino acid play a critical role in those functions are important. In the previous studies, FIPs would cause hemagglutination, and the bio-activity were similar to lectins. Both of them were small proteins with different functions to different cells. In this study, we would like to investigate whether the functions of GMI was related to the binding ability to the glycosylation of cell surface receptors just like lectins. From the result, the immunomodulatory function of GMI would inhibit by adding specific carbohydrates and α-amylase; however, the anti-cancer activity of GMI would not affect. Besides, the aromatic amino acids, especially tryptophan, played a critical role in protein-glycan interactions. Thus, trying to replace the 3 tryptophans of GMI into alanine or glycine to see whether these three amino acids are critical to the functions of GMI. The result showed that only the replacement of three tryptophans in the same tine would affect the anti-cancer activity, and the replacement of the 25th or 110th amino acid would affect the immunomodulatory functions of GMI. Overall, the immunomodulatory functions of GMI seemed relative to its carbohydrate binding ability, and the 25th and 110th amino acid of GMI, tryptophan, somehow played an important role in it.

摘要 III
Abstract V
目錄 VII
表目錄 XII
圖目錄 XIII
第一章前言 1
一、靈芝 1
二、靈芝生理活性成分 2
1. 靈芝多醣體 2
2. 三萜類 2
3. 核酸類 3
4. 真菌免疫調節蛋白質 3
三、小孢子靈芝免疫調節蛋白質 4
1. 蛋白質結構 4
2. 免疫調節活性 5
3. 抗腫瘤活性 5
四、凝集素 (Lectins) 6
1. 動物中的凝集素 6
2. 植物中的凝集素 7
3. 凝集素的應用 7
五、Alanine scanning 8
六、Pichia pastoris表達系統 8
七、研究動機 9
1. 目的 9
2. 策略 10
3. 欲達成目標 11
第二章、材料方法 16
一、 細菌與真菌實驗 16
1. 培養基與藥品 16
2. 菌株與培養條件 18
3. 質體建構與轉形株 19
4. 嗜甲醇酵母菌電穿孔轉形法 21
5. 轉形株篩選 22
二、蛋白質表現與分析 22
1. 蛋白質表現 22
2. 蛋白質純化與濃縮 23
3. 蛋白質定量 24
4. 聚丙烯醯胺膠體電泳 24
5. 西方墨點法 25
三、細胞培養與分析 27
1. 試劑與培養基 27
2. 細胞株及培養條件 27
3. 抗腫瘤活性測試 28
4. 細胞免疫活性測試 29
二、 統計分析 31
第三章、實驗結果 35
一、 嗜甲醇酵母菌表現系統表現免疫調節蛋白質 35
1. 突變質體構築 35
2. 表現菌株篩選 35
3. 蛋白質表現、純化與分析 36
二、 抗腫瘤活性測試 37
1. 醣結合競爭測試 (Sugar binding Competition assay) 37
2. 澱粉酶影響GMI抗腫瘤活性 37
3. 突變型GMI抗腫瘤活性 38
三、 細胞免疫活性測試 38
1. 醣結合競爭測試 (Sugar binding Competition assay) 38
2. 澱粉酶影響GMI免疫調節作用 39
3. 突變型GMI免疫調節作用測定 39
第四章、討論 70
一、 Alanine scanning 70
二、 抗腫瘤活性測試 70
三、 細胞免疫活性測試 71
第五章、總結 73
第六章、未來展望 74
一、 Alanine scanning 74
二、 抗腫瘤活性測試 74
三、 細胞免疫活性測試 74
第七章、參考文獻 76

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