臺灣博碩士論文加值系統

由金針菇(Flammulina Velutipes)表現的免疫調節功能蛋白(Fungal immunomodulatory protein,FIP,)可以使正常人類週邊血液的淋巴球(human peripheral blood lymphocytes) 及對小鼠脾臟細胞誘發干擾素(interferon-γ)增生，此外可以抑制Th2細胞所產生的細胞激素，但金針菇生長週期長且欲從金針菇中分離純化得到高純度之FIP-fve效率不高，每克金針菇可以純化出8.75微克 FIP，因此本研究利用與金針菇同屬真菌之嗜甲基酵母菌為宿主來表現生產FIP，將FIP-fve基因建構於含AOXI啟動子 P.pastoris pPIC9K 質體中，此質體經轉形進入P.pastoris GS115，經篩選確定染色體中含有FIP-fve基因之菌株命名為P. pastoris-AαFIPfve。此菌株用搖瓶培養在30℃、300rpm，培養5天後可得到乾菌重12.83(g/l)，經金屬螯合層析純化後的蛋白質有1.615(mg/g cell)。

Fungal immunomodulatory protein (FIP-fve) can be isolated and purified from Flammulina velutipes with very low yield (8.75μl/g biomass) . FIP-fve contains 114 amino acid residus with an acetylated C-terminal . FIP-fve can stimulate the proliferation of human peripheral blood lymphocytes , induce mice spleen cells to express interferon-γ , and inhibit Th2 cell from producing cytokine like IL-4, IL-5,IL-6,IL-10 and IL-13 . In this thesis methylotrophic yeast Pichia pastoris was employed to express FIP-fve. An integrative vector containing alcohol oxidase (AOX1) promoter controlled FIP-fve gene was contracted and transformed into P. pastoris GS115. Since α-factor prepro signal peptides of the Saccharomyces cerevisiae was employed to direct the secretion of FIP-fve, FIP-fve was detected in the fermentation broth after 1%(v/v) methanol induction. One gram of P. pastoris can produce 1~2 mg FIP-fve after purified by immobilized metal ion affinity chromatography (IMAC). FIP-fve were observed three major bands in SDS-PAGE including 14kDa, 18kDa and 24kDa . In western bolting analysis using either anti-His antibody or anti-FIPfve antibody as probe also detected these three protein bands

中文摘要 …………………………………….…………………..……I

英文摘要 ……………………………….……………………………..II

目錄 …………………………………………………………...…….III

圖目錄 ……………………...………….………………………….VI

第一章、緒論 ……………………………………………..…………….1
1.1前言 ……………………….…………………………..……………1
1.2研究動機 ………………………………...…………....……………4
第二章、文獻回顧 ………………………….…………..…..………..…5
2.1 金針菇免疫調節蛋白(FIP-fve) ……………….………………..5
2.1.1 金針菇 …………………….…………………..…………………5
2.1.2 免疫調節蛋白的研究發展 ………………………….…………6
2.1.2 FIP-fve的生理活性 ……………….…………………………7
2.2 嗜甲基酵母菌Pichia pastoris表現系統 ………………………9
2.2.1 嗜甲基酵母菌Pichia pastoris表現系統的優點 ………..….…9
2.2.2 醣基化作用 ……….………………………….…….…….....….10
2.2.3 P. pastoris的表現宿主菌株 ………………….………...…16
2.2.4 分泌性訊息胜肽 ……………………….………...…………...17


2.2.5 酵母菌基因重組以及轉殖 …………………….…….…......…21
第三章、實驗方法 …………………...………………....…………..…25
3.1 實驗材料 …………….…………………………….…………….25
3.1.1 菌株及來源 ………………………………….…….…….……..25
3.1.2 質體 ……………………………………….…..…..……….…..25
3.1.3 抗體 ……………………….………………...…….….….…….26
3.1.4 質體DNA純化套件組 …...………………..……..……….….26
3.1.5 標準分子量溶液 …………………………..……………….....26
3.1.6 藥品 ………………………….…………..………….…….….. 26
3.1.7 培養基 ………………………..…………...………………....…29
3.2 實驗設備 ……………………………..…………..…………....30
3.3 實驗步驟 ..……..…….……….……...…………………….…..31
3.3.1 利用酵母菌Pichia pastoris GS115 以AOX I 啟動子表現His-tagged FIPfve實驗流程 ….......31
3.3.2 大量複製質體 ………..………………………….……..….…..34
3.3.2.1質體轉殖入宿主細胞(E. coli TOP10 E. coli BL21(DE3)) ………..……..34
3.3.2.2質體的純化 …………………………...………..…...………...35

3.3.3 pPIC9K-AαFIPfve線性化 …………………...….………..…....36
3.3.4 DNA沉澱 ………..…………………..….….…..…….………36
3.3.5 電穿孔轉殖法 …………………………..………...….……..…37
3.3.6 篩選轉殖菌株 …………………………..…....……...……...…38
3.3.7 酵母菌染色體的分離 ………………..…..……..………….…39
3.3.8 pPIC9K-FIPf質體的建構 ………………………...….…….….40
3.3.9 使用搖瓶表現蛋白質 …………………..………….…………41
3.3.10重組蛋白質純化 ……………………..…..…………………...41
3.3.11蛋白質之濃度分析 ………………….………………………...42
3.3.12 蛋白質電泳分析 ………………..……..……………………...43
3.3.13 免疫分析 (Western blot) …………..…..……………………..43
3.3.14 Pichia pastoris的發酵培養表現FIPfve ..................................45
3.3.15 發酵槽操作 …………………….……………..…….………...45
3.3.15.1 pH電極之校正與保存 ……………….………...…………..45
3.3.15.2溶氧(DO)電極之校正與保存 ………………..……..………46
第四章、結果與討論 …………………………..…… ……………….48
4.1 金針菇免疫調節蛋白(FIP-fve)表現菌株之建構 ………....….48
4.2 確認Pichia pastoris所表現的FIP-fve ………….……...….....51

4.3 FIP-fve蛋白質濃度檢測 ………………...…………………..…55
4.4 單體/雙體(Monomer/Dimer)之測定 ……………….………....…57
4.5 Casamino Acid對FIP-fve產量的影響 ……………….………...58
4.6 甲醇添加量對蛋白質表現量的影響 ……………..…....………60
4.7 甲醇添加方式對FIP-fve產量的影響 ..…..………....….….……63
第五章、結論 ………………………………………………………...67
文獻參考 ……………………………………………………………...69

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