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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Yu-Bon Chiang | en |
| dc.contributor.author | 蔣友邦 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:47:07Z | - |
| dc.date.available | 2014-08-10 | |
| dc.date.copyright | 2012-08-10 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-23 | |
| dc.identifier.citation | 1. 許瑞祥 (1988) 靈芝的奧秘 (臺北市).
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(2009) Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens. Vaccine 27 Suppl 6:G21-26. 57. Ooi VE & Liu F (2000) Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Current medicinal chemistry 7(7):715-729. 58. 陳妙齡 (1989) 以松杉靈芝餵食BALB/c鼠探討腹腔免疫反應的功能評估指標. 碩士論文 (國立臺灣大學). 59. Lin CC, et al. (2011) A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells. Cancer immunology, immunotherapy : CII 60(7):1019-1027. 60. Melief CJ (2008) Cancer immunotherapy by dendritic cells. Immunity 29(3):372-383. 61. 盧則豫 (2010) 松杉靈芝三萜類對高油飲食小鼠代謝症候群指標與發炎介質的影響. 碩士論文 (國立臺灣大學). 62. Ko HH, Hung CF, Wang JP, & Lin CN (2008) Antiinflammatory triterpenoids and steroids from Ganoderma lucidum and G. tsugae. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65505 | - |
| dc.description.abstract | 靈芝是中國傳統醫學的珍貴藥材，具有滋補強身之效。近代科學研究也證實靈芝具有抗腫瘤與增強免疫力之功效，而靈芝的藥性成分可分為三萜類、多醣體、真菌免疫調節蛋白質三類。本研究探討選殖自小孢子靈芝Ganoderma microsporum的免疫調節蛋白質GMI，由嗜甲醇酵母菌Pichia pastoris以甲醇誘導方式進行胞外表現重組蛋白質GMI，並利用醱酵槽大規模生產。蛋白質純化後進行免疫活性測試結果顯示GMI能促進T細胞株Jurkat分泌IL-2、活化小鼠骨髓樹突細胞表面蛋白質作抗原呈獻且刺激其分泌IL-12、降低巨噬細胞株Raw264.7因添加LPS造成的促發炎細胞激素分泌量。
GMI由111個胺基酸組成，分子量為12.6 kDa，具高度水溶性。蛋白質於水溶液中的單體間會因N端α-helix形成分子間疏水性作用力及氫鍵，使兩單體結合為同源雙體結構。本篇研究利用點突變方式，將GMI蛋白質N端α-helix的第六個胺基酸，由原本的leucine (L)突變為cycteine (C)、lysine (K)或phenylalanine (F)，藉此探討不同程度雙體化的GMI是否會對免疫活性造成差異，並以膠體電泳和奈米微粒分析儀探討突變型蛋白質的雙體化程度，佐以免疫細胞實驗。結果顯示雙體化比率最高的突變型GMI-L6C，和野生型GMI相比，免疫調節活性顯著提升；而無法形成雙體的突變型組別(GMI-L6K、GMI-L6F)則免疫調節活性下降。由此確認真菌類免疫調節蛋白質雙體化所扮演的角色，提供未來真菌免疫調節蛋白質藥理活性提升的參考。 | zh_TW |
| dc.description.abstract | Lingzhi, a popular medicinal edible mushroom, has been used in Asia for the past 2000 years. The antitumoral and immune-stimulating effects of Lingzhi are attributed to polysaccharides, triterpenoids and fungal immunomodulatory proteins (FIPs). This study investigated GMI, a FIP cloned from Ganoderma microsporum and expressed in a methytrophic yeast, Pichia pastoris. The native form GMI is a noncovalently linked homodimer, and the monomer consists of 111 amino acids, with molecular weight of 12.4 kDa.
In this study, three different point mutation GMIs here made at the sixth residue, from leucine (L) to cysteine (C), lysine (K) and phenylalanine (F) respectively, resulting of the mutant GMIs, GMI-L6C, GMI-L6K and GMI-L6F. These mutants and wild type GMI here used to study the role of dimerization in immune modulatory functions and protein structure. GMI-L6C is able to form a stable homodimer via the formation of a disulfide bond. GMI-L6K and GMI-L6F tend to be pure monomer due to the positive charge or steroic conformation. Based on the results of the immune cell assays and nanoparticle analyzer, homodimer form GMI-L6C is able to increase immune cells function, while the monomer form GMI-L6K and GMI-L6F are unable to enhance immune activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:47:07Z (GMT). No. of bitstreams: 1 ntu-101-R99b22049-1.pdf: 6486827 bytes, checksum: e60043783fde6294b4b92099ebc6eba5 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 目錄 I
圖目錄 IV 表目錄 V 中文摘要 VI ABSTRACT VII 第一章 前言 1 一、 靈芝 1 二、 靈芝藥性成分及生理活性 2 1. 靈芝多醣體 2 2. 三萜類 2 3. 真菌免疫調節蛋白質 3 三、 小孢子靈芝免疫調節蛋白質 5 1. 蛋白質結構 5 2. 免疫調節活性 5 四、 以雙體化程度研究GMI功能 9 五、 蛋白質雙體化判定 10 1. Polyacrylamide gel electrophoresis (PAGE) 10 2. 奈米微粒分析儀 10 六、 Pichia pastoris表達系統 12 七、 免疫系統 13 八、 GMI未來發展 15 九、 研究動機與目的 16 1. 以P. pastoris異源表達重組蛋白質GMI及其突變型 16 2. 探討GMI第六位置胺基酸對雙體化程度之影響 16 3. 比較GMI及其突變型，不同程度雙體化於免疫細胞刺激的差異 16 第二章 材料與方法 18 一、 細菌與真菌實驗 18 1. 培養基與藥品 18 2. 菌株與培養條件 19 二、 質體與轉形株 21 1. pPICZαA-ΔC-myc 21 2. pPICZαA-ΔC-myc-GMI 21 3. pPICZαA-ΔC-myc-L6C 21 4. pPICZαA-ΔC-myc-L6K 21 5. pPICZαA-ΔC-myc-L6F 21 6. 酵母菌轉形株 22 7. P. pastoris異源蛋白質表現 22 三、 醱酵槽大量生產目標蛋白質 25 1. 藥品與儀器 25 2. 培養條件 25 四、 重組蛋白質分析 27 1. 蛋白質濃縮與純化 27 2. 蛋白質定量 28 3. 蛋白質聚丙烯醯胺膠體電泳 28 4. 西方墨點法(Western blot) 29 五、 奈米微粒分析儀 31 六、 細胞培養與細胞激素含量分析 32 1. 試劑與培養基 32 2. 細胞培養條件 32 3. 細胞存活分析 34 4. 細胞激素含量分析 34 七、 統計分析 37 第三章 結果與討論 38 一、 重組蛋白質表現與純化 38 1. 重組蛋白質生產(搖瓶培養) 38 2. 重組蛋白質生產(醱酵槽培養) 41 2. 蛋白質構形分析 43 二、 奈米微粒分析儀 46 三、 免疫細胞刺激 51 1. 人類淋巴癌T細胞株Jurkat 51 2. 小鼠單核球巨噬細胞株 RAW264.7 53 3. 骨髓來源樹突細胞(BMDCs) 55 四、 蛋白質熱穩定性分析 58 第四章 總結 62 第五章 未來展望 63 第六章 參考文獻 65 | |
| dc.language.iso | zh-TW | |
| dc.subject | 奈米微粒分析儀 | zh_TW |
| dc.subject | 免疫細胞 | zh_TW |
| dc.subject | 雙體化 | zh_TW |
| dc.subject | 真菌免疫調節蛋白質 | zh_TW |
| dc.subject | 靈芝 | zh_TW |
| dc.subject | Lingzhi | en |
| dc.subject | fungal immunomodulatory proteins (FIPs) | en |
| dc.subject | dimerization | en |
| dc.subject | nano-particle analyzer | en |
| dc.subject | immune cells | en |
| dc.title | 小孢子靈芝免疫調節蛋白質GMI雙體化對其免疫調節功能之影響 | zh_TW |
| dc.title | Effect of the dimerization of the Ganoderma microsporum protein GMI on its immune-modulatory function | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃介辰(Chieh-Chen Huang),許瑞祥(Ruey-Shyang Hseu),張世宗(Shih-Chung Chang),楊啟伸(Chii-Shen Yang) | |
| dc.subject.keyword | 靈芝,真菌免疫調節蛋白質,雙體化,免疫細胞,奈米微粒分析儀, | zh_TW |
| dc.subject.keyword | Lingzhi,fungal immunomodulatory proteins (FIPs),dimerization,nano-particle analyzer,immune cells, | en |
| dc.relation.page | 72 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-24 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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