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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 許輔 | |
dc.contributor.author | Chih-Liang Hung | en |
dc.contributor.author | 洪志良 | zh_TW |
dc.date.accessioned | 2021-06-15T02:26:31Z | - |
dc.date.available | 2009-08-19 | |
dc.date.copyright | 2009-08-19 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43708 | - |
dc.description.abstract | 本研究以銀耳 (Tremella fuciformis Berk) 免疫調節蛋白 TFP1 為材料,目的在深入探討 TFP1 之生化特性,以及研究 TFP1 對小鼠腹腔巨噬細胞的免疫調節活性與活化路徑,並建立酵母菌的異體表現系統。在探討 TFP1 的生化特性方面,根據質譜儀分析之結果,發現 native TFP1 之分子量約 11.0 kDa,與其基因轉錄後預期的分子量 (11.4 kDa) 相近,為重組 TFP1 經膠體層析與SDS-PAGE 電泳所得值 (約 24.0 kDa) 之半,因此推測 TFP1 在自然環境下可能為同質雙元體。在研究 TFP1 調節免疫的作用方面,實驗發現 TFP1 能夠活化小鼠腹腔巨噬細胞產生 TNF- a 及 IL-1b,同時也能提高 TNF-a, IL-1b, IL-6, IL-12p35, IL-12p40, CCL3, 以及 CXCL10 的 mRNA 表現,同時流式細胞分析發現 TFP1 可增加腹腔巨噬細胞吞噬能力及CD86 之表現,且電泳位移分析 (EMSA assay) 顯示 TFP1 能夠活化 NF-kB 轉錄因子;另一方面,TFP1 活化小鼠腹腔巨噬細胞的作用,會受 anti-TLR2/TLR4 抗體中和處理以及 TLR4-/- 基因缺陷所抑制,故推測 TFP1 活化腹腔巨噬細胞的作用與 TLR2/TLR4 活化 NF-kB 之路徑高度相關。另一方面,將 TFP1 基因轉殖置入 pYEX-S1 載體,於 Saccharomyces cerevisiae 酵母菌株 DY150 表現得到重組 TFP1及帶有 His-tag 的 TFP1。本研究之結果進一步釐清 TFP1 活化巨噬細胞的機轉,並有助於將 TFP1 開發成飼料或調節免疫產品。 | zh_TW |
dc.description.abstract | TFP1 is an immunomudulatory protein previously purified from jelly fungi (Tremella fuciformis Berk). The objectives of this study was to clarify the biochemical characteristics of TFP1, to further investigate its regulating function and its activation pathway on mouse peritoneal macrophages as well as to construct its yeast expression system. Mass spectrometry analyses obtained the molecular weight of native TFP1 (11.0 kDa), agreeing its genetically translated value (11.3 kDa). Based on gel-filtration and SDS-PAGE analyses, the recombinant TFP1 showed a molecular mass of 24 kDa, suggesting that TFP1 could be a homodimer protein. In addition, TFP1 activated mouse peritoneal macrophages, increased the production of TNF-a and IL-1b and enhanced the mRNA expression of TNF-a, IL-1b, IL-6, IL-12p35, IL-12p40, CCL3, and CXCL10. Flow cytometry analysis demonstrated that TFP1 increased the phagocytosis activity and CD86 expression by the cells. Moreover, EMSA result showed a TFP1-induced activation of the transcription factor NF-kB. The stimulatory effects of TFP1-promoted TNF-a secretion on macrophages were inhibited under antibody neutralization and TLR4 deficiency (TLR4-/-). Based on these results, TLR2/TLR4 signaling pathway was suggested to be involved in TFP1-induced NF-kB activation. Furthermore, TFP1 gene was cloned into pYEX-S1 vector and expressed by Saccharomyces cerevisiae to produce recombinant TFP1 and His-tagged TFP1. This study clarified the mechanism and activities of TFP1 on mouse peritoneal macrophages and also provided more information on the application of feed and food utilization. | en |
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dc.description.tableofcontents | 口試委員會審定書
謝誌 中文摘要...........................................................................................................................1 英文摘要...........................................................................................................................2 第一章 研究背景.............................................................................................................3 第一節 前言..............................................................................................................3 第二節 食藥用菇類的保健功效..............................................................................3 一、菇類生理活性.............................................................................................4 二、真菌免疫調節蛋白.....................................................................................6 第三節 銀耳之生理特徵..........................................................................................7 一、銀耳之簡介.................................................................................................7 二、銀耳之型態特性.........................................................................................8 三、銀耳之化學成分組成.................................................................................8 第四節 已知銀耳生理活性物質與作用..................................................................9 一、抗腫瘤功效.................................................................................................9 二、降血脂功效...............................................................................................10 三、調節免疫活性.............................................................................................10 四、降血糖活性................................................................................................11 五、銀耳免疫調節蛋白之發現與研究............................................................11 第五節 巨噬細胞的作用........................................................................................12 第六節 酵母菌蛋白質異體表現系統....................................................................14 第七節 研究動機與架構........................................................................................18 第二章 材料與方法.......................................................................................................19 第一節 銀耳免疫調節蛋白 TFP1 之製備...........................................................19 一、TFP1 粗萃取..............................................................................................19 二、快速蛋白質液相層析系統分析 (Fast Protein Liquid Chromatography).20 三、SDS-PAGE 膠體電泳...............................................................................22 四、透析及濃縮................................................................................................24 五、蛋白質濃度測定........................................................................................25 第二節 免疫調節活性試驗....................................................................................26 一、腹腔巨噬細胞取得.....................................................................................26 二、細胞激素 ELISA 測定法.........................................................................29 三、腹腔巨噬細胞吞噬活性分析.....................................................................30 四、腹腔巨噬細胞抗原呈現能力分析.............................................................33 五、腹腔巨噬細胞 mRNA 萃取.....................................................................35 六、反轉錄聚合酶連鎖反應 (reverse transcript PCR, RT-PCR) ....................36 七、即時定量聚合酶連鎖反應 (real-time quantitative PCR) .........................37 八、電泳位移分析 (electrophoretic mobility shift assay, EMSA) ..................39 第三節 TFP1 活化巨噬細胞路徑.........................................................................44 第四節 以酵母菌系統表現重組銀免疫調節蛋白 TFP1 ....................................46 一、酵母菌表現載體構築...............................................................................46 二、酵母菌轉形作用.......................................................................................49 三、酵母菌菌落聚合酶連鎖反應...................................................................51 四、酵母菌菌株之保存...................................................................................52 五、酵母菌表現 dTFP1 蛋白........................................................................52 六、酵母菌表現 dTFP1 蛋白之檢測.............................................................53 第五節 統計分析....................................................................................................53 第三章 結果...................................................................................................................54 第一節 分析 TFP1 的生化特徵...........................................................................54 一、蛋白質分子量分析...................................................................................54 二、胺基酸序列 signal peptide 預測.............................................................54 三、預測 TFP1 可能醣化形式及位置...........................................................55 四、預測 TFP1 蛋白質二級結構...................................................................55 五、TFP1 胺基酸序列同源性比對.................................................................56 第二節 TFP1 之純化.............................................................................................57 第三節 TFP1 對小鼠腹腔巨噬細胞之活化作用.................................................59 一、TFP1 可增加細胞激素 TNF-a 與 IL-1b 之分泌................................59 二、TFP1 可增加小鼠腹腔巨噬細胞之吞噬能力..........................................60 三、TFP1 對小鼠腹腔巨噬細胞呈獻抗原能力之影響..................................60 四、TFP1 對小鼠腹腔巨噬細胞之細胞激素 mRNA 表現之影響...............61 五、TFP1 對小鼠腹腔巨噬細胞極化之影響..................................................62 第四節 TFP1 對NF-kB 轉錄因子之影響以及 TLR 路徑相關性...................63 一、TFP 1 可活化 nuclear factor kappa B (NF-kB) 轉錄因子......................63 二、TFP1 可能透過 TLR4 以及 TLR2 受體活化小鼠腹腔巨噬細胞.......64 第五節 利用酵母菌系統表現重組 TFP1.............................................................65 一、重組 TFP1 基因片段之取得及 insert 之構築.......................................65 二、製備無外泌序列之表現載體....................................................................66 三、pYEX構築表現載體 pYEX-DTFP1 及 pYEX-His-tag-DTFP1.............66 四、在酵母菌株 DY150 表現重組 TFP1 及重組 His-tag TFP1.................67 第四章 討論...................................................................................................................69 第五章 結論...................................................................................................................73 參考文獻.........................................................................................................................74 圖表.................................................................................................................................85 附錄...............................................................................................................................110 圖表目錄 Fig 1. Scheme of the study...............................................................................................85 Fig 2. MALDI-TOF analysis of TFP1.............................................................................86 Fig 3. SignalP 3.0 predictions of TFP1 ...........................................................................87 Fig 4. YinOYang 1.2 Predicition Results of TFP1...........................................................88 Fig 5. NetNGlyc 1.0 prediction results of TFP1 .............................................................89 Fig 6. The secondary structure of TFP1, predicted by GOR method .............................90 Fig 7. Alignment of amino acid sequence of TFP1, extensin family protein (Ef; Laccaria bicolor S238N-H82), and riboflavine-aldehyde-forming enzyme (Raf; Laccaria bicolor S238N-H82) .........................................................................91 Fig 8. Preparation of TFP1..............................................................................................92 Fig 9. Stimulatory effects of TFP1 on the production of TNF-α and IL-1b by mouse peritoneal macrophages.....................................................................................93 Fig 10. Stimulatory effects of TFP1 on the phagocytic activity in mouse peritoneal macrophages......................................................................................................94 Fig 11. Effects of TFP1 on the expression of CD80 in mouse peritoneal macrophages.95 Fig 12. Stimulatory effects of TFP1 on the expression of CD86 in mouse peritoneal macrophages......................................................................................................96 Fig 13. Effects of TFP1 on the expression of MHC class II in mouse peritoneal macrophages......................................................................................................97 Fig 14. TFP1 up-regulates TNF-a, IL-1b, and IL-6 mRNA expressions in mouse peritoneal macrophages in a time-dependent manner........................................98 Fig 15. TFP1 up-regulates M1 type chemokines mRNA expression in mouse peritoneal macrophages in a time-dependent manner. .......................................................99 Fig 16. TFP1 up-regulates M2 type chemokines mRNA expression in mouse peritoneal macrophages in a time-dependent manner......................................................100 Fig 17. Effects of TFP1 on the mRNA expressions of IL-10 and IL-12 in mouse peritoneal macrophages.................................................................................................101 Fig 18. Expression of NF-kB in mouse peritoneal macrophages exposed to TFP1.....102 Fig 19. Expression of NF-kB in mouse peritoneal macrophages exposed to TFP1 at different treatment times..................................................................................103 Fig 20 . Effects of TFP1 on the mRNA expressions of TLR2 and TLR4 in mouse peritoneal macrophages.................................................................................................104 Fig 21. TFP1 activated mouse peritoneal macrophages via TLR2 and TLR4..............105 Fig 22. The procedure for constructing expression plasmid..........................................106 Fig 23. Map of pYEX-DTFP1 expression vector..........................................................107 Fig 24. Determination of recombinant TFP1 from yeast transformant.........................108 Table 1. The result of protein-protein Blast analysis for TFP1.....................................109 附錄 Appendix 1. Real-time PCR primers used in this study................................................110 Appendix 2. SDS-PAGE analysis (A) and Periodic acid/Schiff’s staining (B) showed that TFP1 and TFP2 are not glycoproteins...............................................111 Appendix 3. HPLC-MALS analysis of TFP1................................................................112 Appendix 4. Complete nucleotide sequence of TFP1 cDNA and its deduced amino acid sequence...................................................................................................113 Appendix 5. TFP1 N-terminal amino acid sequence.....................................................114 Appendix 6. TFP1 cDNA sequence in GeneBank®......................................................115 Appendix 7. TFP1 amino acid sequence in GeneBank®..............................................116 Appendix 8. Map of pYEX-S1 vector...........................................................................117 | |
dc.language.iso | zh-TW | |
dc.title | 銀耳TFP1蛋白對小鼠腹腔巨噬細胞免疫調節作用及異體表現之研究 | zh_TW |
dc.title | Investigation of the Immunomodulatory Effects of the TFP1 Protein from Tremella Fuciformis Berk. on Mouse Peritoneal Marcophages and its Heterologus Expression | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐源泰,周志輝,劉?睿 | |
dc.subject.keyword | 銀耳,免疫調節蛋白,腹腔巨噬細胞,酵母菌,異體表現, | zh_TW |
dc.subject.keyword | Tremella fuciformis Berk,immunomudulatory protein,peritoneal macrophages,yeast,heterologus expression, | en |
dc.relation.page | 117 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-18 | |
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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