請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45967完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Ming-I Lai | en |
| dc.contributor.author | 賴明毅 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:50:03Z | - |
| dc.date.available | 2015-08-10 | |
| dc.date.copyright | 2010-08-10 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-02 | |
| dc.identifier.citation | 1. Agaphonov MO, Beburov M, Ter-Avanesyan MD, Smirnov VN: A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha. Yeast 11: 1241-7 (1995).
2. Bogdanova AI, Agaphonov MO, Ter-Avanesyan MD: Plasmid reorganization during integrative transformation in Hansenula polymorpha. Yeast 11: 343-53 (1995). 3. Boh B, Berovic M, Zhang J, Zhi-Bin L: Ganoderma lucidum and its pharmaceutically active compounds. In: El-Gewely MR (ed) Biotechnology Annual Review, pp. 265-301. Elsevier (2007). 4. Cereghino G, Sunga A, Cereghino J, Cregg J: Expression of Foreign Genes in the yeast Pichia pastoris, pp. 157-169 (2001). 5. Cereghino JL, Cregg JM: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24: 45-66 (2000). 6. Chang H-H, Hsieh K-Y, Yeh C-H, Tu Y-P, Sheu F: Oral administration of an Enoki mushroom protein FVE activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma. International Immunopharmacology 10: 239-246 (2010). 7. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA: Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9: 1316-23 (1989). 8. Daly R, Hearn MT: Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18: 119-38 (2005). 9. Davies DH: Immune System. Encyclopedia of Life Sciences - Immunology: 1-10 (2008). 10. Demain AL, Vaishnav P: Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances 27: 297-306 (2009). 11. Ding Y, Seow SV, Huang CH, Liew LM, Lim YC, Kuo IC, Chua KY: Coadministration of the fungal immunomodulatory protein FIP-Fve and a tumour-associated antigen enhanced antitumour immunity. Immunology 128: e881-94 (2009). 12. Durum SK: Cytokines and the Immune System Cytokine Reference, pp. 95-97. 13. Faber KN, Haima P, Gietl C, Harder W, Ab G, Veenhuis M: The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins). Proc Natl Acad Sci U S A 91: 12985-9 (1994). 14. Faber KN, Haima P, Harder W, Veenhuis M, Ab G: Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet 25: 305-10 (1994). 15. Fehervari Z, Sakaguchi S: T Lymphocytes: Regulatory. Encyclopedia of Life Sciences - Immunology: 1-8 (2008). 16. Gatzke R, Weydemann U, Janowicz ZA, Hollenberg CP: Stable multicopy integration of vector sequences in Hansenula polymorpha. Appl Microbiol Biotechnol 43: 844-9 (1995). 17. Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E, Veenhuis M, Klei I: New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison. FEMS Yeast Research 5: 1079-1096 (2005). 18. Gellissen PDG, Strasser AWM, Suckow M: Key and Criteria to the Selection of An Expression Platform. In: Prof. Dr. Gerd G (ed) Production of Recombinant Proteins, pp. 1-5 (2005). 19. Gray D, Subramanian S: Choice of cellular protein expression system. Curr Protoc Protein Sci Chapter 5: Unit5 16 (2001). 20. Haak-Frendscho M, Kino K, Sone T, Jardieu P: Ling Zhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol 150: 101-13 (1993). 21. Hartner FS, Glieder A: Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact 5: 39 (2006). 22. Hiep TT, Noskov VN, Pavlov YI: Transformation in the methylotrophic yeast Pichia methanolica utilizing homologous ADE1 and heterologous Saccharomyces cerevisiae ADE2 and LEU2 genes as genetic markers. Yeast 9: 1189-1197 (1993). 23. Honeyman M, Harrison L: Immunity: Humoral and Cellular. Encyclopedia of Life Sciences - Immunology: 1-10 (2004). 24. Hsieh CW, Lan JL, Meng Q, Cheng YW, Huang HM, Tsai JJ: Eosinophil apoptosis induced by fungal immunomodulatory peptide-fve via reducing IL-5alpha receptor. J Formos Med Assoc 106: 36-43 (2007). 25. Hsieh KY, Hsu CI, Lin JY, Tsai CC, Lin RH: Oral administration of an edible-mushroom-derived protein inhibits the development of food-allergic reactions in mice. Clin Exp Allergy 33: 1595-602 (2003). 26. Hsu HC, Hsu CI, Lin RH, Kao CL, Lin JY: Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem J 323 ( Pt 2): 557-65 (1997). 27. Hsu HY, Hua KF, Wu WC, Hsu J, Weng ST, Lin TL, Liu CY, Hseu RS, Huang CT: Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells. J Cell Physiol 215: 15-26 (2008). 28. Huang L, Sun F, Liang C, He YX, Bao R, Liu L, Zhou CZ: Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium. Proteins 75: 524-7 (2009). 29. Kelley LA, Sternberg MJE: Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protocols 4: 363-371 (2009). 30. Kino K, Mizumoto K, Sone T, Yamaji T, Watanabe J, Yamashita A, Yamaoka K, Shimizu K, Ko K, Tsunoo H: An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice. Diabetologia 33: 713-8 (1990). 31. Kino K, Sone T, Watanabe J, Yamashita A, Tsuboi H, Miyajima H, Tsunoo H: Immunomodulator, LZ-8, prevents antibody production in mice. Int J Immunopharmacol 13: 1109-15 (1991). 32. Kino K, Yamashita A, Yamaoka K, Watanabe J, Tanaka S, Ko K, Shimizu K, Tsunoo H: Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. J Biol Chem 264: 472-8 (1989). 33. Ko HH, Hung CF, Wang JP, Lin CN: Antiinflammatory triterpenoids and steroids from Ganoderma lucidum and G. tsugae. Phytochemistry 69: 234-9 (2008). 34. Ko JL, Hsu CI, Lin RH, Kao CL, Lin JY: A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem 228: 244-9 (1995). 35. Lahtchev K: Basic Genetics of Hansenula polymorpha. In: Gerd G (ed) Hansenula polymorpha, pp. 8-20 (2004). 36. Lin-Cereghino J, Lin-Cereghino GP: Vectors and strains for expression. Methods Mol Biol 389: 11-26 (2007). 37. Lin WH, Hung CH, Hsu CI, Lin JY: 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: 20044-8 (1997). 38. Lin YL, Liang YC, Tseng YS, Huang HY, Chou SY, Hseu RS, Huang CT, Chiang BL: An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways. J Leukoc Biol 86: 877-89 (2009). 39. Liu YH, Kao MC, Lai YL, Tsai JJ: Efficacy of local nasal immunotherapy for Dp2-induced airway inflammation in mice: Using Dp2 peptide and fungal immunomodulatory peptide. J Allergy Clin Immunol 112: 301-10 (2003). 40. Lukic ML, Lukic AM: Immune Response: Regulation. Encyclopedia of Life Sciences - Immunology: 1-6 (2002). 41. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM: Heterologous protein production using the Pichia pastoris expression system. Yeast 22: 249-70 (2005). 42. Mahajna J, Dotan N, Zaidman BZ, Petrova RD, Wasser SP: Pharmacological values of medicinal mushrooms for prostate cancer therapy: the case of Ganoderma lucidum. Nutr Cancer 61: 16-26 (2009). 43. Middelhoven WJ: History, Habitat, Variability, Nomenclature and Phylogenetic Position of Hansenula polymorpha. In: Gerd G (ed) Hansenula polymorpha, pp. 1-7 (2004). 44. Nakagawa T, Inagaki A, Ito T, Fujimura S, Miyaji T, Yurimoto H, Kato N, Sakai Y, Tomizuka N: Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica. Yeast 23: 15-22 (2006). 45. Nakagawa T, Mizumura T, Mukaiyama H, Miyaji T, Yurimoto H, Kato N, Sakai Y, Tomizuka N: Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica. Yeast 19: 1067-1073 (2002). 46. O’Shea JJ, Nutman TB: Immunoregulation. Encyclopedia of Life Sciences - Immunology: 1-10 (2001). 47. Ou CC, Hsiao YM, Wu WJ, Tasy GJ, Ko JL, Lin MY: FIP-fve stimulates interferon-gamma production via modulation of calcium release and PKC-alpha activation. Journal of Agricultural & Food Chemistry 57: 11008-13 (2009). 48. Paaventhan P, Joseph JS, Seow SV, Vaday S, Robinson H, Chua KY, Kolatkar PR: A 1.7A structure of Fve, a member of the new fungal immunomodulatory protein family. J Mol Biol 332: 461-70 (2003). 49. Raschke WC, Neiditch BR, Hendricks M, Cregg JM: Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris. Gene 177: 163-7 (1996). 50. Raymond CK, Bukowski T, Holderman SD, Ching AF, Vanaja E, Stamm MR: Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase. Yeast 14: 11-23 (1998). 51. Rodriguez L, Narciandi RE, Roca H, Cremata J, Montesinos R, Rodriguez E, Grillo JM, Muzio V, Herrera LS, Delgado JM: Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris. Yeast 12: 815-22 (1996). 52. Sanodiya BS, Thakur GS, Baghel RK, Prasad GB, Bisen PS: Ganoderma lucidum: a potent pharmacological macrofungus. Curr Pharm Biotechnol 10: 717-42 (2009). 53. Seow SV, Kuo IC, Paaventhan P, Kolatkar PR, Chua KY: Crystallization and preliminary X-ray crystallographic studies on the fungal immunomodulatory protein Fve from the golden needle mushroom (Flammulina velutipes). Acta Crystallogr D Biol Crystallogr 59: 1487-9 (2003). 54. Sheu F, Chien P-J, Hsieh K-Y, Chin K-L, Huang W-T, Tsao C-Y, Chen Y-F, Cheng H-C, Chang H-H: Purification, Cloning, and Functional Characterization of a Novel Immunomodulatory Protein from Antrodia camphorata (Bitter Mushroom) That Exhibits TLR2-Dependent NF-κB Activation and M1 Polarization within Murine Macrophages. Journal of Agricultural and Food Chemistry 57: 4130-4141 (2009). 55. Siverio JM: Biochemistry and Genetics of Nitrate Assimilation. In: Gerd G (ed) Hansenula polymorpha, pp. 21-40 (2004). 56. Sliva D: Ganoderma Lucidum(Reishi) in Cancer Treatment. Integr Cancer Ther 2: 358-364 (2003). 57. Sliva D: Cellular and physiological effects of Ganoderma lucidum (Reishi). Mini Rev Med Chem 4: 873-9 (2004). 58. Tanaka S, Ko K, Kino K, Tsuchiya K, Yamashita A, Murasugi A, Sakuma S, Tsunoo H: 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: 16372-7 (1989). 59. van der Heide M, Leao AN, Van der Klei IJ, Veenhuis M: Redirection of peroxisomal alcohol oxidase of Hansenula polymorpha to the secretory pathway. FEMS Yeast Res 7: 1093-102 (2007). 60. van der Hem LG, van der Vliet JA, Bocken CF, Kino K, Hoitsma AJ, Tax WJ: Ling Zhi-8: studies of a new immunomodulating agent. Transplantation 60: 438-43 (1995). 61. van der Klei IJ, Hilbrands RE, Kiel JA, Rasmussen SW, Cregg JM, Veenhuis M: The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery. EMBO Journal 17: 3608-18 (1998). 62. van Dijk R, Faber KN, Kiel JA, Veenhuis M, van der Klei I: The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26: 793-800 (2000). 63. Viguera A-R, Serrano L: Loop length, intramolecular diffusion and protein folding. Nat Struct Mol Biol 4: 939-946 (1997). 64. Wang H, Lu F, Sun Y, Du L: Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica. Biotechnology Letters 26: 1569-73 (2004). 65. Wang PH, Hsu CI, Tang SC, Huang YL, Lin JY, Ko JL: Fungal immunomodulatory protein from Flammulina velutipes induces interferon-gamma production through p38 mitogen-activated protein kinase signaling pathway. J Agric Food Chem 52: 2721-5 (2004). 66. Weikl TR: Loop-closure principles in protein folding. Archives of Biochemistry and Biophysics 469: 67-75 (2008). 67. Wu CC, Liao EC, Lee MF, Tsai JJ: Augmentation of regulatory T cells in allergic individuals by recombinant Der f 2 peptide with fungal immunomodulatory peptide fve. Ann Allergy Asthma Immunol 102: 216-22 (2009). 68. Wu S, Letchworth GJ: High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36: 152-4 (2004). 69. Yeh CM, Yeh CK, Hsu XY, Luo QM, Lin MY: Extracellular expression of a functional recombinant Ganoderma lucidium immunomodulatory protein by Bacillus subtilis and Lactococcus lactis. Appl Environ Microbiol 74: 1039-49 (2008). 70. Yuen JWM, Gohel MDI: Anticancer Effects of Ganoderma lucidum: A Review of Scientific Evidence. Nutrition and Cancer 53: 11 - 17 (2005). 71. Zhou X, Lin J, Yin Y, Zhao J, Sun X, Tang K: Ganodermataceae: natural products and their related pharmacological functions. Am J Chin Med 35: 559-74 (2007). 72. 吳明玥: 利用熱休克蛋白質5'端非轉譯區片段調控免疫調節蛋白質GMI於米麴菌表達系統之產量. 國立臺灣大學微生物與生化學研究所碩士論文 (2008). 73. 李啟睿: 利用漢遜酵母MOX及FMD啟動子異源表現瘤胃真菌Neocallimastix frontalis之木聚醣酶. 國立臺灣大學微生物與生化學研究所碩士論文 (2006). 74. 林千椀: 利用分子演化增加小孢子靈芝選殖之免疫調節蛋白質GMI的免疫活性. 國立臺灣大學微生物與生化學研究所碩士論文 (2008). 75. 林采蔆: 靈芝屬免疫調節蛋白GMI與GFO-1基因之選殖與Pichia pastoris之異源表現. 國立臺灣大學微生物與生化學研究所碩士論文 (2005). 76. 張文馨: 真菌類免疫調節蛋白 FIP-vvo 作用機制之研究. 國立臺灣大學醫學院生物化學暨分子生物學研究所碩士論文 (2004). 77. 莊榮輝: 水稻蔗糖合成酶之研究. 國立臺灣大學農業化學研究所博士論文 (1985). 78. 許瑞祥: 靈芝概論. 萬年, 臺中市 (1993). 79. 陳長志: 大腸桿菌植酸酶於重組酵母菌Pichia pastoris中大量表現之研究. 國立臺灣大學微生物與生化學研究所博士論文 (2005). 80. 黃玉儒: 靈芝免疫調節蛋白的作用機制及其減緩塵蟎致敏之應用. 國立中山醫學大學毒理學研究所碩士論文 (2003). 81. 蔡孟男: 利用甘油受限誘導漢遜氏酵母菌表現重組蛋白質. 國立臺灣大學微生物與生化學研究所碩士論文 (2008). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45967 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:50:03Z (GMT). No. of bitstreams: 1 ntu-99-R97B47408-1.pdf: 8035243 bytes, checksum: f1f54fbe955a51e1339858dee9eed88e (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 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 | |
| dc.language.iso | zh-TW | |
| dc.subject | 嗜甲醇酵母菌 | zh_TW |
| dc.subject | 真菌免疫調節蛋白質 | zh_TW |
| dc.subject | 免疫調節功能 | zh_TW |
| dc.subject | immune modulation | en |
| dc.subject | Fungal immunomodulatory proteins | en |
| dc.title | 以嗜甲醇酵母菌表現靈芝屬免疫調節蛋白質LZ-8、GMI和GFO-1並探討其免疫功能 | zh_TW |
| dc.title | Overexpression of LZ-8, GMI and GFO-1, the immunomodulatory proteins from Ganoderma spp. in methylotrophic yeasts and investigation of their immune functions | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許瑞祥,陳浩仁,楊啟伸,陳俊任 | |
| dc.subject.keyword | 真菌免疫調節蛋白質,免疫調節功能,嗜甲醇酵母菌, | zh_TW |
| dc.subject.keyword | Fungal immunomodulatory proteins,immune modulation, | en |
| dc.relation.page | 116 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 未授權公開取用 | 7.85 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。