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研究生: 吳宗勳
Wu, Tsung-Hsun
論文名稱: 茯苓表現序列標誌基因庫之建構分析與免疫調節蛋白基因之選殖
Construction and analysis of EST library, and cloning of genes encoding immunomodulatory proteins in Wolfiporia cocos
指導教授: 孫智雯
Sun, Chih-Wen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2005
畢業學年度: 93
語文別: 中文
論文頁數: 65
中文關鍵詞: 茯苓肌動蛋白基因庫免疫調節蛋白
英文關鍵詞: Wolfiporia cocos, actin, EST library, FIP
論文種類: 學術論文
相關次數: 點閱:342下載:25
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  • 許多的食、藥用蕈類含有多種固醇類、三類、核酸、多醣等藥用活性或潛力的成分,具有抗腫瘤與增進免疫力的功能。而近年的研究發現茯苓(Wolfiporia cocos)與樟芝(Taiwanofungus camphorate)的蛋白質亦具免疫調節活性,亦可歸為真菌免疫調節蛋白(fungal immunomodulatory proteins, FIPs)。茯苓是一種在中藥方劑裡應用範圍最廣的真菌。然而,其生物活性成份有關知識仍有限,且基因體研究尚在起步階段。因此,本研究欲自茯苓菌絲體中尋找可調節免疫系統功能的蛋白質,並建立一個茯苓表現序列標誌(express sequence tag, EST)基因庫。在分析液態培養茯苓菌絲體cDNA基因庫的過程中,發現一個與樟芝免疫調節蛋白(Aca1)胺基酸序列相似的基因(相似度達50%)。透過表現載體轉殖到大腸桿菌中,經誘導表現所純化出來的茯苓與樟芝重組蛋白質,皆能被茯苓蛋白單株抗體辨識。推測此蛋白質,是可能的茯苓免疫調節蛋白的成員之一。

    Most edible and medical fungi contain ingradents, such as steroids, triterpenes, nuclear acids, and polysaccharides. These compounds are commonly considered to have pharmaceutical activities or potentials, and therefore function in arresting the growth of tumer cells or stimulating immune systems.
    Few proteins have recently isolated from Wolfiporia cocos and Taiwanofungus camphorate with immunomodulatory activity, and are described as fungal immunomodulatory proteins (FIPs). Wolfiporia cocos is the most popular fungal ingredient in Chinese prescription, however, knowledge regarding to its bioactive compounds and genomic information is limited. Thus, I would like to explore FIP candidates, and establish a express sequence tag (EST) library from Wolfiporia cocos in this study. A potential FIP gene was characterized from 1-week-old Wolfiporia cocos cDNA library. This gene encodes a protein sharing 50% sequence similarity to Taiwanofungus camphorate FIP (Aca1), and is therefore named as WcFIP1. The recombinant WcFIP1and Aca1 was expressed in E. coli, and their identity were confirmed by western analysis. We concluded that the newly isolated WcFIP1 is one of the fungal immunomodulatory proteins in Wolfiporia cocos.

    目錄 摘要 3 壹、 緒論 5 第一節 蕈類的免疫調節、抗腫瘤活性 5 第二節 蕈類具免疫調節活性的蛋白質 6 凝集素 7 真菌免疫調節蛋白 7 第三節 茯苓 8 第四節 茯苓成分與藥效 9 第五節 研究目的與問題 9 貳、 材料與方法 11 一、 茯苓菌絲體 11 (一) 固態培養茯苓菌絲體 11 (二) 液態培養茯苓菌絲體 11 二、 茯苓RNA/ mRNA的萃取、合成1st-cDNA 12 (一) total RNA 12 (二) LiCl沉澱法 12 (三) Agarose凝膠電泳 13 (四) RNA的定量 13 (五) mRNA的萃取 14 (六) 合成首股(1st-strand) cDNA 15 三、 選殖肌動蛋白(Actin)、確認RNA與cDNA品質 15 (一) 設計引子擴增茯苓肌動蛋白 16 (二) 1st-strand cDNA PCR 16 (三) PCR產物選殖 16 (四) 序列比對與專一性引子的設計 18 (五) 茯苓肌動蛋白3’-RACE 18 (六) 利用RT-PCR確認cDNA、RNA 19 四、 茯苓免疫調節蛋白的選殖 19 (一) 退化性引子(degenerated primer) 19 (二) RT-PCR 19 五、 cDNA library 20 (一) 建構cDNA library 20 (二) 基因序列分析比對 21 六、 建構表現重組蛋白載體 21 (一) 設計寡核酸引子 21 (二) 構築全長WcFIP1於表現載體 22 (三) BL21(DE3)pLysS轉形勝任細胞的製備 22 (四) JM109(DE3)pLysS轉形勝任細胞的製備 23 (五) 熱休克法轉形表現宿主(expression host) 24 (六) 轉形JM109(DE3)pLysS 24 七、 表現重組蛋白 25 (一) 以IPTG誘導重組蛋白表現 25 (二) 裂解細胞 26 (三) 回溶重組蛋白 27 (四) 純化重組蛋白 27 (五) SDS-PAGE 28 (六) CBBR染色 29 (七) 西方轉漬 29 八、 活性測試 32 參、 結果 33 茯苓RNA的萃取及cDNA的合成與肌動蛋白(Actin)選殖 33 茯苓免疫調節蛋白基因的選殖 34 茯苓cDNA基因庫的建構 34 茯苓免疫調節蛋白WcFIP1的特性 35 WcFIP1重組蛋白的純化 36 肆、 討論 37 茯苓免疫調節蛋白的重要性 37 茯苓肌動蛋白的選殖 37 茯苓免疫調節蛋白的選殖 38 基因表現序列分析 38 茯苓免疫調節蛋白的分析 39 抑制細菌生長 40 總結 40 伍、 誌謝 40 陸、 參考文獻 41 柒、 圖表 46

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