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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:322下載:17
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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

    Adachi, K., H. Nanba, and H. Kuroda, 1987. Potentiantion of host-mediated antitumor activity in mice by beta-glucan obtained from Grifola frondosa (maitake). Chem. Pharm. Bull. (Tokyo). 35:262-270.
    Adams, M. D, 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185-2195.
    Ainsworth, G. C., F. K. Sparrow, and A. S. Sussman, 1973. The fungi. Ⅳ. A taxonomic review with keys: Ascomycetes and fungi imperfecti, Academic press, Inc. New York.
    Benson, D. A., I. Karsch-Mizrachi, and D. J. Lipman, J. Ostell, B. A. Rapp, and D. L. Wheeler, 2000. GenBank. Nucl. Acids Res. 28(1):15-18
    Bernard, S. J., I. Yuan, C. McMillen, M. E. Symonds, P. C. Owens, 1999. Abundance of lepin mRNA in fetal adipose tissue is related to fetal body weight. Journal of Endocrinology 163(1), R1-4
    Blattner, F. R., et al., 1997. The complete genomic sequence of Escherichia coli K12. Science 277: 1453-1462.
    Chang, S., J. Puryear, and J. Cairney, 1993. A Simple and Efficient Method for isolating RNA from Pine Trees. Plant Molecular Biology Reporter 11: 113-116.
    Cheung, V. G., et al., 2001. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409: 953 – 958.
    Collins, R. A. and T. B. Ng, 1997. Polysaccharopeptide from Coriolus versicolor has potential for use against human immunodeficiency virus type 1 infection. Life Sci. 60(25): PL383-7.
    Debray, H., D. Dus, P. Husso, C. Radzikowski, and J. Montreuil, 1990. Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Aftered glycosylation of membrane glycoproteins. Chn. Exp. Metastasis. 8: 287-298.
    Ennamany, R, O. Kretz, A. Badoc, G. Deffieux, E. E. Creppy, 1994. Effect of bolesatine, a glycoprotein from Boletus satanas, on rat thymus in vivo. Toxicol. 89: 113-118.
    Granger, G. A. and T. W. Williams, 1968. Lymphocyte cytotoxicity in vitro: activation and release of a cytotoxic factor. Nature 218:1253-1254
    Hall, N., J. P. R. Keon, and J. A. Hargreaves, 1999. A homologue of a gene implicated in the virulence of human fungal diseases is present in a plant fungal pathogen and is expressed during infection. Physiological and Molecular Plant Pathology 55: 69-73.
    Hattori, T., K. Hayashi, T. Nagao, K. Furuta, M. Ito, and Y. Suzuki, 1992. Studies on antinephritic effects of plant components (3): Effect of pachyman, a main component of Poria cocos Wolf on original-type anti-GBM nephritis in rats and its mechanisms. Jpn. J. Pharmacol. 59(1):89-96.
    Hibino, Y., Y. Konishi, J. Koike, T. Tabata, Y. Ohashi, and N. Sugano, 1994. Productions of interferon-gamma and nitrite are induced in mouse splenic cells by a heteroglycan-protein fraction from culture medium of Lentinus edodes mycelia. Immunopharm. 28(1):77-85.
    Hikino, H., 1985. Recent research on oriental medicinal plants. In: Wanger, H., H. Hikino, and N.R. Farnsworth(Eds), Ecnomic and medicinal plant research. 1. Acaemic Press, London. p64-65.
    Hseu, Y. C, W. C. Chang, Y. T. Hseu, C. Y. Lee, Y. J Yech, P. C. Chen, J. Y. Chen, and H. L. Yang 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sci. 71(4):469-82.
    Hsu HC, Hsu CI, Lin RH, Kao CL, Lin LY. 1997. Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem. J. 323 ( Pt 2): 557-565.
    Ito, H., K. Shimura, H. Itoh, and M. Kawade, 1997. Antitumor effects of a new polysaccharide-protein complex (ATOM) prepared from Agaricus blazei (Iwade strain 101) "Himematsutake" and its mechanisms in tumor-bearing mice. Anticancer Res. 17(1A):277-284.
    Inoue A, Kodama N, and Nanba H., 2002. Effect of maitake (Grifola frondosa) D-fraction on the control of the T lymph node Th-1/Th-2 proportion. Biol Pharm Bull. 25(4):536-40.
    Jeune, K. H., L. J. Moon, M. K. Kim, and S. R. Chung, 1990. Studies on lectins from korean higher fungi; IV. A mitogenic lectin from the mushroom Lentinus edodes. Plants Med. 56: 592.
    Jin, Y., L. Zhang, M. Zhang, L. Chen, P. C. Cheung, V. E. Oi, and Y. Lin, 2003. Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media. Carbohydr. Res. 338(14):1517-21.
    Jong, S. C., J. M. Birmingham, and S. H. Pai, 1991. Immunomodulatory substances of fungal origin. J. Immuno. Immunopharmacol. 11: 115-122.
    Kaminaga T., K. Yasukawa, H. Kanno, T. Tai, Y. Nunoura, and M. Takido, 1996. Inhibitory effects of lanostane-type triterpene acids, the components of Poria cocos, on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Oncology 53(5): 382-5.
    Kanayama, H., N. Adachi, and M. Togami, 1983. A new antitumor polysaccharide from the mycelia of Poria cocos wolf. Chem. Pharm. Bull. (Tokyo) 31(3): 1115-8.
    Kanehisa, M., S.Goto, S. Kawashima, Y. Okuno, and M. Hattori, 2004. The KEGG resources for deciphering the genome. Nucleic Acids Res. 32, D277-D280.
    Kaul, S., et al., 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815.
    Kim, K. S., K. H. Kim, S. E Choi, J.-W. Yoon, and Y. Kang, 1999. Soluble expression in Escherichia coli of murine endogenous retroviral transmembrane envelope protein having immunosuppressive activity. Protein Expression and Purification 16, 19–26.

    Kino, K., A. Yamashita, K. Yamaoka, J. Watanabe, S. Tanaka, K. Ko, K. Shimizu, and H. Tsunoo, 1989. Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. J. Biol. Chem. 264(1): 472-428.
    Kino, K., T. Sone, J. Watanabe, A. Yamashita, H. Tsuboi, H. Miyajima, and H. Tsunoo, 1991. Immunomodulator, LZ-8, prevents antibody production in mice. Int. J. Immunopharmacol. 13(8): 1109-1115.
    Ko, J. L., C. I. Hsu, R. H. Lin, C. L. Kao, and L. Y. Lin. 1995. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur. J. Biochem. 228(2): 244-249.
    Ko, J. L., S. J. Lin, C. I. Hsu, C. L. Kao, and L. Y. Lin. 1997. Molecular cloning and expression of a fungal immunomodulatory protein, FIP-fve, from Flammulina velutipes. J. Formos. Med. Assoc. 96(7): 517-524.
    Kobayashi, Y., K. Kariya, K. Saigenji, and K. Nakamura, 1994. Suppression of cancer cell growth in vitro by the protein-bound polysaccharide of Coriolus versicolor QUEL (PS-K) with SOD mimicking activity. Cancer Biother. 9(1): 63-9.
    Khler, G, and C.Milstein, 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495.
    Lee, I. H., R. L. Huang, C. T. Chen, H. C. Chen, W. C. Hsu, and M. K. Lu, 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett. 209(1):63-7.
    Lee, S. S., Y. H. Wei, C. F. Chen, S. Y. Wang, and K. Y. Chen, 1995. Antitumor effects of Ganoderma lucidum. J. Chinese Med. 6:1-12.
    Licastro, F., M. C. Morini, O. Kretz, G. Dirheimer, E. E. Creppy, and F. Stirpe, 1993. Mitogenic activity and immunological properties of bolesatine, a lectin isolated from the mushroom Boletus satanas Lenz. Int. J. Biochem. 25: 789-792.
    Lieu, C. W., S. S., Lee and S.Y. Wang, 1992. The effect of Ganoderma lucidum on induction of differentiation in leukemic U937 cells. Anticancer Res. 12(4): 1211-1215.
    Lin WH, Hung CH, Hsu CI, Lin JY. 1997. Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J. Biol. Chem. 272(32): 20044-20048.
    Lin, J. Y., and T. B. Chou 1984. Isolation and characterization of a lectin from edible mushroom, Volvariella volvacea. J. Biochem. 96: 35-40.
    Liu, F., V. E. Ooi, and S. T. Chang, 1997. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci. 60(10): 763-771.
    Liu, W. K., T. B. Ng, S. F. Sze, and K.W. Tsui, 1993. Activation of peritoneal macrophages by polysaccharopeptide from the mushroom, Coriolus versicolor. Immunopharmacol. 26(2):139-46.
    Miyasaka, N., H. Inoue, T. Totsuka, R. Koike, K. Kino, H. Tsunoo. 1992. An immunomodulatory protein, Ling Zhi-8, facilitates cellular interaction through modulation of adhesion molecules. Biochem. Biophys. Res. Commun. 186(1):385-390.
    Murasugi, A., S. Tanaka, N. Komiyama, N. Iwata, K. Kino, H. Tsunoo, and S. Sakuma, 1991. Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidum. J. Biol. Chem. 266(4):2486-2493.
    Ng, T. B., 1998. A review of research on the protein-bound polysaccharide (polysaccharopeptide, PSP) from the mushroom Coriolus versicolor (Basidiomycetes: Polyporaceae). Gen Pharmacol. 30(1):1-4.
    Ohsawa, T., M. Yukawa, C. Takao, M. Murayama, and H. Bando, 1992. Studies on constituents of fruit body of Polyporus umbellatus and their cytotoxic activity. Chem. Pharm. Bull. (Tokyo). 40(1):143-7.
    Pajtasz, E., D. Dus, and H. Debray, 1988. Mitogenic stimulation of lymphoid cells by Aleuria aurantia agglutinin. Arch. Invnunol. Exp. Ther. 36: 133-139.
    Sakagami, H., K. Sugaya, A. Utsumi, S. Fujinaga, T. Sato, and M. Takeda, 1993. Stimulation by PSK of interleukin-1 production by human peripheral blood mononuclear cells. Anticancer Res. 13(3): 671-675.
    Schonor, E. G., L. F. Ellis, , and B. E. Schonor, (1985) Isolation and purification of protein granules from E. coli cells overproducing bovine growth hormone. BioTechnology 3, 151–154.
    Tai, T., Y. Akita, K. Kinoshita, K. Koyama, K. Takahashi, and K. Watanabe, 1995. Anti-emetic principles of Poria cocos. Planta Med. 61(6):527-30.
    Takeyama, T, I. Suzuki, N. Ohno, S. Oikawa, K. Sato, M. Ohsawa, and T.Yadomae, 1987. Host-mediated antitumor effect of grifolan NMF-5N, a polysaccharide obtained from Grifola frondosa. J. Pharmacobiodyn. 10(11):644-51.
    Tamura, R., K. Tanebe, C. Kawanishi, K. Torii, and T. Ono, 1997. Effects of lentinan on abnormal ingestive behaviors induced by tumor necrosis factor. Physiol. Behav. 61(3): 399-410.
    Tanaka, S., K. Ko, K. Kino, K. Tsuchiya, A. Yamashita, A. Murasugi, S. Sakuma, and H. Tsunoo, 1989. Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions. J. Biol. Chem. 264(28): 16372-16377.
    Tsang, K. W., C. L. Lam, C. Yan, J. C. Mak, G. C. Ooi, J. C. Ho, B. Lam, R. Man, J. S. Sham, and W. K. Lam, 2003. Coriolus versicolor polysaccharide peptide slows progression of advanced non-small cell lung cancer. Respir Med. 97(6):618-24.
    Tseng, J., and J. G. Chang, 1992. Suppression of tumor necrosis factor-α, interleukin-1 β, interleukin-6 and granulocyte-monocyte colony stimulating factor secretion from human monocytes by an extract of Poria cocos. Chin. J. Microbiol. Immunol. 25(1):1-11.
    Tsuda, M., 1979. Purification and characterization of a lectin from the mushroom Flammulina velutipes. J. Biochem. 86:1463-1468
    van der Hem, L. G., J. A. van der Vliet, K. Kino, A. J. Hoitsma, and W. J. Tax, 1996. Ling-Zhi-8: a fungal protein with immunomodulatory effects. Transplant Proc. 28(2): 958-959.
    Wang, H. X., J. Q. Gao, and T. B. Ng, 2000. A New Lectin with Highly Potent Antihepatoma and Antisarcoma Activities from the Oyster Mushroom Pleurotus Ostreatus. Biochem. Biophys. Res. Commun. 275(3), 810–816
    Wang, H. X., T. B. Ng, W. K. Liu, V. E. Ooi, and Chang S. T., 1995. Isolation and characterization of two distinct lectins with antiproliferative activity from the cultured mycelium of the edible mushroom Tricholoma mongolicum. Int. J. Peptide Protein Res. 46: 508-513.
    Wang, H. X., W. K. Liu, T. B. Ng, V. E. Ooi, and S. T. Chang, 1996. The immuno-modulatory and antitumor activities of lectins from the mushroom Tricholoma mongolicum. Immunopharmacol. 31: 205-211.
    Wasser, S. P. and A. L. Weis, 1999. Therapeutic effects of substances occurring in higher Basidiomycetes mushroom: a modern perspective. Crit. Rev. Immuno. 19: 65-96.
    Wolf, F. A., 1922. Poria cocos. Journal of the Elisha Mitchell Scientific Society 38: 134
    Wood, V., et al., 2002. The genome sequence of Schizosaccharomyces pombe. Nature 415: 871 – 880.
    Yan, S. C., 1988. Linical and experimental research on Polyporus umbellatus polysaccharide in the treatment of chronic viral hepatitis. Zhong Xi Yi Jie He Za Zhi. 8(3):141-3, 131.
    You, J. S., D. M. Hau, K. T. Chen, and H. F. Huang, 1994. Combined effects of chuling (Polyporus umbellatus) extract and mitomycin C on experimental liver cancer. Am. J. Chin. Med. 22(1):19-28.
    Yu, L. G., D. J. Fernig, J. A. Smith, J. D. Milton, and J. M. Rhodes, 1993. Reversible inhibition of proliferation of epithelial cell lines by Agaricus bisporus (edible mushroom) lectin. Cancer Res. 53: 4627-4632.
    Zhang, J., G. Wang, H. Li, C. Zhuang, T. Mizuno, H. Ito, H. Mayuzumi, H. Okamoto, and J. Li, 1994. Antitumor active protein-containing glycans from the Chinese mushroom songshan lingzhi, Ganoderma tsugae mycelium. Biosci. Biotechnol. Biochem. 58(7):1202-1205.
    Zhang, Y. H., Y. L. Liu, S. C. Yan., 1991. Effect of Polyporus umbellatus polysaccharide on function of macrophages in the peritoneal cavities of mice with liver lesions. Zhong Xi Yi Jie He Za Zhi. 11(4):225-6, 198.
    丁懷謙, 2000. 食藥用菇多醣體之免疫生理活性, 食品工業 32:28-42.
    王伯徹, 1990. 藥用真菌系列報導(四). 茯苓. 食品工業22(5): 32-38.
    王伯徹, 2000. 具開發潛力食藥用菇介紹. 食品工業 32: 1-17.
    白壽雄 與 羅道蘊, 1994. 生物性多醣體及其應用. 生物產業 5: 167-173.
    李時珍, 1590. 本草綱目. 卷二十八.
    孫孝洪, 1992. 中醫治療學原理. 知音出版社, 臺北, pp.276.
    徐錦堂, 1997. 中國藥用真菌學. 聯合出版社 pp.547-573.
    財團法人食品工業發展研究所, 2000. 真菌目錄. 財團法人食品工業發展研究所 pp. 161-182
    高正釗, 1985. 新編中藥大辭典(中). 新文豐出版社 pp.1594
    張慧欣, 2005. 茯苓免疫調節蛋白的純化、機能活性及生理角色之探討. 國立台灣大學, 園藝學研究所碩士論文.
    曹巧吟, 2003. 樟芝中免疫調節蛋白的純化與其生理活性之探討. 國立台灣大學, 園藝學研究所碩士論文.
    陳清農, 陳勁初, 張基煌, 與 喬長誠, 1999. 樟芝揮發性成分之研究. 中華民國食品科學技術學會第二十九次大會論文摘要.
    閔三弟, 1996. 真菌的藥用價值. 食用菌學報3(4): 55-64.
    劉波, 1981. 中國藥用真菌, 山西人民出版社 pp.119-120.
    錢家樂, 2004. 樟芝免疫調節蛋白基因選殖及表現之研究. 國立台灣大學, 園藝學研究所碩士論文.
    戴郁軌 與 朱凱俊, 1982. 真菌名詞辭典. 名山出版社 pp.2467.

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