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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許輔 | |
| dc.contributor.author | An-Ju Chang | en |
| dc.contributor.author | 張安如 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:26:15Z | - |
| dc.date.available | 2008-07-26 | |
| dc.date.copyright | 2006-07-26 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | Abbas, A. K. and A. H. Lichtman (2004). 'Basic Immunology,' 2/Ed. W.B. Saunders Company.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33133 | - |
| dc.description.abstract | 白木耳 (Tremella fuciformis Berk) 為重要食藥用菇類之一，本研究以白木耳乾燥子實體為材料，經硫酸銨沈澱及 DE-52 陰離子交換管柱純化出具有免疫調節活性的蛋白區分物 TFPs，進一步以 HiTrap Q 陰離子交換層析與膠體過濾層析將 TFPs 分離出兩個蛋白，分別命名為TFP1 與 TFP2，其分子量分別約為 24 kDa與 40 kDa，以等電聚焦電泳分析發現兩者之 pI 值皆為 3.45。TFP1 和 TFP2 經過醣蛋白染色試驗後，發現兩者皆非醣蛋白，經過血球凝集試驗後，發現兩者皆不具有凝集小鼠紅血球之活性。經過體外免疫活性試驗發現白木耳蛋白 TFP1 與TFP2 均可直接活化 RAW264.7 巨噬細胞分泌 nitric oxide 與 TNF-α (p ＜ 0.05)，此外，TFP1 與 TFP2 也能夠刺激小鼠脾臟細胞增生，而TFP1蛋白在100℃ 或 -80℃ 處理後皆能保持其刺激巨噬細胞分泌 NO 的免疫活性。此外，以細胞融合技術建立 3 株能夠辨識白木耳蛋白 TFPs 之單株抗體，有利於日後之純化與檢測，以及進行相關的蛋白質體學之研究。另一方面以白木耳菌絲體為原料，根據 TFP1 蛋白 N 端胺基酸序列合成 TFP1 之 degenerate primers，嘗試以3’ 及5’ rapid amplification cDNA ends (RACE) 的方法針對 TFP1 進行 cDNA 選殖，但未取得符合序列之基因；由二維電泳與西方轉漬分析發現用來做為原料基因選殖的菌絲體可能沒有表現目標蛋白或表現量很低，遂使得選殖工作遭到困境。序列比對分析各選殖片段產物後發現，白木耳菌絲體的基因可能與 Cryptococcus neoformans var. neoformans JEC21的基因有高度相關性。綜合本研究結果得知，TFP1 與 TFP2 是白木耳中重要的免疫調節活性物質，可提升宿主相關免疫反應，極具醫藥以及臨床發展之潛力。 | zh_TW |
| dc.description.abstract | Tremella fuciformis Berk is not only an edible mushroom but also known to possess pharmacological properties. The new immunomodulatory protein TFPs were purified from the dried fruiting body of T. fuciformis by ammonium sulfate precipitation and DE-52 anion-exchange chromatography. Proteins from the peak fractions were collected and further purified using HiTrap Q ion-exchange and gel filtration chromatography. Finally, two distinct proteins, TFP1 and TFP2 were obtained. SDS-PAGE analysis showed that the molecular weights of TPF1 and TFP2 were 24 kDa and 40 kDa, respectively. Isoelectric focusing electrophoresis revealed that the pI values of both proteins were both approximately 3.45. Both TFP1 and TFP2 were not glycoproteins and none of them could agglutinate mouse red blood cells. In addition, TFP1 and TFP2 could directly activate RAW264.7 macrophages and enhance the production of both nitric oxide (NO) and tumor necrosis factor-alpha (p＜0.05). TFP1 and TFP2 were also capable to stimulate the proliferation of murine splenocytes in vitro. TFP1 remained fairly good activities on stimulating the secretion of NO by RAW264.7 macrophages after different temperature treatments such as 100℃ and -80℃. Moreover, three monoclonal antibodies which recognize TFPs specifically were established, and they could be utilized for purification and other proteomic studies in the future. Furthermore, cDNA cloning of TFP1 protein from the mycelium of T. fuciformis was also performed. TFP1 degenerate primers based on the N-terminal amino acid sequence were made, and 3’ rapid amplification of cDNA ends (RACE) and 5’ RACE PCR were carried out in order to clone the full-length TFP1 cDNA. However, no sequence matched TFP1 was obtained. Results from two-dimensional electrophoresis and Western blotting analysis suggested that TFP1 gene might be expressed at a low level or even not expressed in the mycelium of T. fuciformis, which might contribute to the failure of gene cloning. BLASTX sequence analysis showed that there was a high correlation between T. fuciformis and Cryptococcus neoformans var. neoformans JEC21 genes. This study suggested that TFP1 and TFP2 were important immunomodulatory components in T. fuciformis and could strengthen the immunity of the host. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:26:15Z (GMT). No. of bitstreams: 1 ntu-95-R93628214-1.pdf: 2698683 bytes, checksum: 3c77e05643919dfc5134795647db0a79 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 圖表目錄 III
摘要 1 ABSTRACT 2 第一章 研究背景與動機 4 第一節 菇類植物藥用潛力 4 第二節 菇類機能性蛋白及其生理活性 5 一、 親醣蛋白 (lectins) 5 二、 菇類免疫調節蛋白 (fungal immunomodulatory proteins) 9 三、 醣蛋白凝集素 (agglutinative glycoproteins) 11 第三節 白木耳的介紹 12 一、 外型及分類 12 二、 生長條件 12 三、 商業栽種 13 四、 化學成分組成 14 五、 生理機能 14 第四節 基因選殖策略 16 一、 利用 degenerated primer 選殖 16 二、 同源性蛋白之序列保守區設計引子選殖 18 三、 免疫篩檢法 (immunoscreening) 18 四、 Rapid Amplification of cDNA ends (RACE) 技術於基因選殖之利用 19 第五節 研究動機與架構 20 第二章 材料與方法 22 第一節 白木耳免疫調節蛋白之純化 22 第二節 白木耳蛋白生化特性之分析 25 第三節 白木耳蛋白免疫調節活性試驗 31 第四節 建立白木耳免疫調節蛋白之單株抗體 38 第五節 白木耳免疫調節蛋白基因之選殖 45 第六節 TFP1蛋白於白木耳菌絲體與子實體之差異表現 54 第七節 統計分析 61 第三章 結果 62 第一節 白木耳蛋白之純化及生化特性 62 一、 白木耳子實體之蛋白純化 62 二、 快速蛋白質層析系統 62 三、 膠體電泳分析 (SDS-polyacrylamide slab gel electrophoresis) 63 四、 醣蛋白染色分析 (Periodic acid-Schiff stain) 63 五、 等電點電泳分析 64 六、 血球凝集活性分析 64 第二節 免疫調節活性試驗 64 一、 對巨噬細胞的影響 64 二、 對小鼠脾臟細胞的影響 67 三、 不同溫度處理對TFP1免疫活性之影響 67 四、 LAL 試驗 68 第三節 白木耳免疫調節蛋白單株抗體之製作 69 一、 單株抗體細胞株之製作 69 二、 陽性融合細胞株之篩選 70 三、 單株抗體之生產 70 四、 西方轉漬分析 71 第四節 白木耳免疫調節蛋白TFP1之基因選殖與序列比對 72 一、 白木耳免疫調節蛋白TFP1之N端胺基酸序列分析 72 二、 白木耳菌絲體 RNA 之抽取 74 三、 TFP1 degenerate 3’ primer與5’ primer之設計 74 四、 RACE PCR 76 五、 RACE產物序列比對結果 79 第五節 白木耳免疫調節蛋白TFP1於不同世代之差異表現 80 一、 2D電泳分析 80 二、 西方轉漬分析 80 第四章 討論 81 一、 白木耳蛋白生化特性 81 二、 白木耳蛋白之免疫調節活性 81 三、 白木耳蛋白之單株抗體建立 82 四、 白木耳免疫調節蛋白TFP1之基因選殖 83 五、 序列分析與比對 84 六、 結論 85 REFERENCE: 87 | |
| dc.language.iso | zh-TW | |
| dc.subject | 基因選殖 | zh_TW |
| dc.subject | 白木耳 | zh_TW |
| dc.subject | 免疫調節蛋白 | zh_TW |
| dc.subject | Tremella fuciformis | en |
| dc.subject | immunomodulatory protein | en |
| dc.subject | gene cloning | en |
| dc.title | 白木耳中免疫調節蛋白活性分析與基因選殖之研究 | zh_TW |
| dc.title | Functional Analysis and Molecular Cloning of Immunomodulatory Protein from Tremella fuciformis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 繆希椿,何國傑,沈湯龍 | |
| dc.subject.keyword | 白木耳,免疫調節蛋白,基因選殖, | zh_TW |
| dc.subject.keyword | Tremella fuciformis,immunomodulatory protein,gene cloning, | en |
| dc.relation.page | 144 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-22 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝學研究所 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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