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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Tsai-Leng Lin | en |
| dc.contributor.author | 林采蔆 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:46:07Z | - |
| dc.date.available | 2007-08-01 | |
| dc.date.copyright | 2005-08-01 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-29 | |
| dc.identifier.citation | 王璽凱，2004。金頂側耳中免疫調節蛋白的純化與活性評估。國立臺灣大學園藝學研究所碩士論文。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35268 | - |
| dc.description.abstract | 靈芝自古便是中國傳統醫學中的珍貴藥材，被認為具有滋補強身、扶正固本之效。近代科學研究也證實靈芝萃取物具有抗腫瘤與增強免疫力之功能，然而多數研究認為靈芝主要活性成分為多醣體及三萜類化合物，直至 1989 年 Kino 等人自靈芝（Ganoderma lucidum）菌絲體純化出具有免疫調節功能之小分子蛋白質 LZ-8，確立另一種活性物質免疫調節蛋白之存在。
本研究針對十種靈芝屬菌株進行聚合酶連鎖反應分析，發現靈芝免疫調節蛋白 LZ-8 相似基因普遍存在於靈芝屬菌株，然而這些菌株所得之基因序列並非皆與lz-8相同，部分菌株更發現同時具有兩種以上的 LZ-8 相似基因。此外採用 genome walking 技術於 G. microsporum 及 G. fornicatum 分別選殖出三條靈芝屬免疫調節蛋白新基因 gmi、gfo-1 與 gfo-2，其中以 gfo-2 與 lz-8 序列相似度最低且序列長度亦不同，gfo-1 則與 lz-8 有較高之序列相似度。 將 lz-8、gmi 及 gfo-1 等基因送入 Pichia pastoris KM71 以甲醇誘導方式進行胞外表現，可得重組蛋白 rePLZ、reGMI 及 reGFO-1，MALDI-TOF 分析結果顯示重組蛋白並無任何醣基化現象，最後目標蛋白產量平均可達 300 mg/l culture。甲醇誘導方式使菌體在需氧量以及 H2O2 累積上易造成壓力，因此重組蛋白 rePLZ、reGMI 及 reGFO-1 於表現後期發生蛋白酶水解現象，且此等蛋白酶無法以親和管柱純化方式去除，但 EDTA 與 pepstatin A 等蛋白酶抑制劑可完全抑制重組蛋白之降解。 rePLZ、reGMI 與 reGFO-1 於胺基酸序列的差異反映在蛋白質構形上，西方雜合結果顯示 reGFO-1 與 rePLZ 於構形上具有較高之相似度，而 reGMI 之構形則與 rePLZ 相差較大。rePLZ、reGMI 及 reGFO-1 進行免疫調節活性測定，皆可刺激 BALB/c 老鼠骨髓之樹突細胞（dendritic cells, DCs）分泌 IL-12，也可刺激老鼠巨噬細胞株 J774A.1 分泌 TNF-α 及刺激人類 T 細胞株 Jurkat cells 分泌 IL-2。其中，reGMI 於 5 μg/ml 下可刺激 DCs 分泌 IL-12 的量為相同濃度 rePLZ 之六倍。 | zh_TW |
| dc.description.abstract | Ganoderma lucidum has been used in Chinese medical application for a long time. Several studies reported that G. lucidum exhibited anti-tumor and immunomodulatroy activities. Polysaccharides and triterperoid were regarded as the major bioactive substances. Another bioactive material was not found until the small protein LZ-8 was purified from mycelium of G. lucidum in 1989.
In this study, LZ-8 primers were used to amplify ten strains of Ganoderma spp. genomic DNA. LZ-8-like sequences were universally found in Ganoderma spp. strains. These LZ-8-like sequences were not exactly identical to LZ-8. Some strains contained two different LZ-8-like sequences. Three new genes, gmi, gfo-1 and gfo-2, were successfully cloned from G. microsporum and G. fornicatum. Three genes, lz-8, gmi and gfo-1, were expressed extracellularly in Pichia pastoris KM71 with methanol induction. No glycosylation in recombinant proteins rePLZ, reGMI and reGFO-1 was found after MALDI-TOF analysis. The average productivities could achieve 300 mg/l culture. Proteolysis occurred at late stage of expression because of oxygen and H2O2 stress induced by methanol. Proteases could not be removed after purification but could be inhibited by EDTA and pepstatin A completely. The differences in protein conformation between rePLZ, reGMI and reGFO-1 was attributed to their amino acid sequences. reGFO-1 was conformationally similar to rePLZ while reGMI was different from rePLZ. Bone marrow derivative dendritic cells from BALB/c mice were stimulated by rePLZ, reGMI and reGFO-1 to secrete IL-12. The concentration of IL-12 stimulated by reGMI was five times higher than that by rePLZ. rePLZ, reGMI and reGFO-1 stimulated murine macrophage J774A.1 cells to secrete TNF-α and Jurkate cells（human T cell line）to secrete IL-2. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T06:46:07Z (GMT). No. of bitstreams: 1 ntu-94-R92b47402-1.pdf: 1578230 bytes, checksum: 609f50d3c84a81d7e245d56b4f9adf12 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 目 錄
表目錄 IV 圖目錄 V 摘 要 VII Abstract IX 第一章 前言 1 一、 靈芝 1 二、 靈芝免疫調節蛋白 3 三、 異源表現系統 8 四、 Pichia pastoris 表現系統 10 五、 研究動機與目的 14 第二章 材料與方法 17 ㄧ、 菌株、質體與引子 17 二、 實驗方法 22 1. 靈芝屬免疫調節蛋白之選殖 22 1.1. 靈芝菌株之篩選 22 1.2. 靈芝染色體 DNA 之萃取 22 1.3. 聚合酶連鎖反應（PCR）分析 24 1.4. Genome Walking 24 1.4.1 模版 DNA 之製備 24 1.4.2 聚合酶連鎖反應 26 2. 靈芝免疫調節蛋白 LZ-8 於 E. coli 之異源表現 28 2.1. pGEXL 表現載體之建構 28 2.2. E. coli融合蛋白之表現 28 2.3. E. coli融合蛋白之純化 28 3. 靈芝屬免疫調節蛋白於 P. pastoris 之異源表現 30 3.1. pPLZ、pPGMI 及 pPGFO-1表現載體之建構 30 3.2. P. pastoris 電穿孔轉形法 30 3.3. P. pastoris 融合蛋白之表現 32 3.4. P. pastoris融合蛋白之純化 32 4. P. pastoris 組蛋白質之特性分析 33 4.1. 蛋白質定量 33 4.2. 西方雜合分析（Western blot hybridization） 33 4.3. MALDI-TOF（Matrix-Assisted Laser Desorption Ionization Time-of-Flight）分析 34 4.4. 蛋白酶抑制劑分析 34 4.5. 單體/雙體（Monomer / Dimer）之測定 34 4.6. 免疫調節活性測試 35 4.6.1. 樹突細胞（Dendritic cell） 35 4.6.2. 巨噬細胞（Macrophage）與 T 細胞 35 第三章 結果 36 一、靈芝屬免疫調節蛋白之選殖 36 1.聚合酶連鎖反應（PCR）分析 36 2. Genome Walking 40 二、靈芝免疫調節蛋白 LZ-8 於 E. coli 異源表現 50 三、靈芝屬免疫調節蛋白於 Pichia pastoris 異源表現 52 1. P. pastoris 融合蛋白之表現 52 2. P. pastoris 融合蛋白之純化 54 四、 Pichia pastoris 重組蛋白質之特性分析 56 1. 西方雜合分析 56 2. MALDI-TOF 分析 58 3. 蛋白酶抑制劑分析 60 4. 單體/雙體（Monomer / Dimer）之測定 62 5. 免疫調節活性測試 64 第四章 討論 66 ㄧ、靈芝屬（Ganoderma spp.）免疫調節蛋白 66 二、靈芝免疫調節蛋白之異源表現 67 第五章 結論 71 參考文獻 72 附圖 76 | |
| dc.language.iso | zh-TW | |
| dc.subject | 免疫調節蛋白 | zh_TW |
| dc.subject | 靈芝 | zh_TW |
| dc.subject | Ganoderma | en |
| dc.subject | Immunomodulatory protein | en |
| dc.subject | Pichia pastoris | en |
| dc.title | 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 | zh_TW |
| dc.title | Gene cloning of immunomodulatory proteins, GMI and GFO-1 from Ganoderma spp. and expression in Pichia pastoris | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張煥正,江伯倫,林璧鳳,許瑞祥 | |
| dc.subject.keyword | 免疫調節蛋白,靈芝, | zh_TW |
| dc.subject.keyword | Immunomodulatory protein,Ganoderma,Pichia pastoris, | en |
| dc.relation.page | 80 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-29 | |
| 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 | |
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