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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Wen-Hsu Ho | en |
| dc.contributor.author | 何文旭 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:11:42Z | - |
| dc.date.copyright | 2006-08-09 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23848 | - |
| dc.description.abstract | Aspergillus屬表達系統目前已廣泛地應用於工業蛋白質生產，特別是A. oryzae等高產量菌株。然而，此表達系統面臨最大的瓶頸是異源蛋白質產量過低的問題。近年來，已發展出一些可以提升異源蛋白質產量的策略，包括建立蛋白質水解酶缺陷突變株、使用強力啟動子、基因融合策略、利用5’端非轉譯區（5’UTR）調控轉譯效率，以及調控蛋白陪伴分子（chaperone）表現量以提升異源蛋白質之分泌等。
本研究利用上述策略中的強力啟動子與基因融合策略，搭配電穿孔轉形系統以米麴菌異源表達由小孢子靈芝（Ganoderma microsporum）選殖之免疫調節蛋白質GMI，並利用缺乏C端澱粉結合區的葡萄澱粉酶（GLaA1-511）以基因融合方式將目標蛋白質分泌至胞外。結果共篩選到2株GLA::GFP與6株GLA::GMI轉形株，經南方氏雜合分析結果顯示質體插入染色體的套數為1至多個不等。由西方雜合法得知異源蛋白質確實可以藉由基因融合策略分泌至胞外。此外，融合蛋白質斷裂的現象可能發生在胞內或培養基中，而且以固態基質培養的分離效果較佳。另外，附著於GFP轉形株菌絲邊緣水珠發出綠色螢光的現象，可能是胞內GFP蛋白質透過非古典分泌途徑分泌到胞外所造成。此外，純化後得到的重組蛋白質GMI可以活化人類T細胞，促使其分泌介白素2（IL-2），顯示米麴菌表達系統生產的GMI具有免疫調節活性。 關鍵字：米麴菌、表達系統、基因融合、GMI、免疫調節蛋白質、電穿孔轉形法。 | zh_TW |
| dc.description.abstract | Aspergillus expression system is generally used for the production of industrial proteins, especially those high yield species, such as A. oryzae. However, the main bottleneck of this system is the low yield for heterologous protein expression. In recent years, several strategies have been developed for improving the heterologous protein production, including the use of protease-deficient strains, strong promoters, gene fusions and 5’UTR for improving the translation efficiency as well as the modulation of chaperone gene expression for improving the secretion.
In this study, we used the strong promoter and gene fusion strategies in conjunction with electroporation to express GMI, an immunomodulatory protein cloned from Ganoderma microsporum in A. oryzae. We also investigated the feasibility of secreting the target proteins using a truncated glucoamylase gene（glaA1-511）fusion. After electroporation, two GLA::GFP and six GLA::GMI transformants were selected. Southern blot analysis of transformants showed that the target genes were integrated into chromosome by single or multiple copies. In addition, the heterologous proteins were secreted into medium by gene fusion strategy, and fusion proteins might be cleaved in cytoplasm or medium. Drops with green fluorescence on the filaments of GFP transformant might result from GFP secretion via non-classical secretion pathway. The immunoactivity analysis of recombinant GMI indicated that human Jurkat T cell could be stimulated to secrete IL-2 by recombinant GMI. Keywords：Aspergillus oryzae, expression system, gene fusion, GMI, immunomodulatory protein, electroporation | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:11:42Z (GMT). No. of bitstreams: 1 ntu-95-R93b47403-1.pdf: 1831651 bytes, checksum: f0a8ecb294a03318cdecb7d0d6dd58f9 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄 I
表目錄 IV 圖目錄 V 摘要 VI Abstract VII 第一章、前言 1 一、基因轉形概述與異源表達 1 1. 常見之微生物表達系統 2 1.1 原核表達系統 2 1.1.1 大腸桿菌 2 1.1.2 枯草桿菌 2 1.2 真核表達系統 3 1.2.1 酵母菌 3 1.2.2 絲狀真菌 4 1.2.3 食用菇類 8 二、麴菌屬（Aseprgillus spp.）表達系統 9 1. 選殖標誌（selection marker） 9 1.1 營養缺陷標誌（autotrophic marker） 9 1.2 抗藥性標誌（antibiotic-resistance marker） 10 2. 轉形策略（transformation strategy） 12 2.1 原生質體（protoplast）PEG轉形法 12 2.2 基因槍（biolistic）轉形法 16 2.3 農桿菌（Agrobacterium-mediated）轉形法 18 2.4 電穿孔（electroporation）轉形法 20 3. 增加異源蛋白質產量之策略 23 3.1 強力啟動子（strong promoter） 24 3.2 利用5’端非轉譯區（5’ untranslated region）調控轉譯效率 26 3.3 基因融合策略（gene fusion strategy） 27 3.4 調控蛋白陪伴分子（chaperone）表現以提升異源蛋白質之分泌 30 3.5 蛋白質水解酶缺陷突變株（protease-deficient strain）之建立 32 三、免疫調節蛋白質 34 1. 靈芝 34 2. 免疫調節蛋白質LZ-8與GMI 35 四、研究動機與目的 37 第二章、材料與方法 40 一、實驗材料 40 1. 菌株與培養條件 40 1.1 真菌 40 1.2 細菌 41 2. 質體 41 2.1 pPTRI 41 2.2 pPGT 41 2.3 pPGFT/pPGAFT 42 2.4 pPGMT/PGAMT 42 二、實驗方法 44 1. 質體建構 44 1.1 pPGFT 44 1.2 pPGMT 45 1.3 pPGAMT 45 1.4 pPGAFT 46 2. 米麴菌之pyrithiamine耐受度測試 51 2.1米麴菌孢子之取得 51 2.2 Pyrithiamine耐受度測試 51 3. 電穿孔轉形 51 3.1 質體DNA之製備 51 3.2 轉形材料之製備 52 3.2.1 孢子萌發處理 52 3.2.2 酵素溶液製備 52 3.2.3 酵素處理 52 3.3 電穿孔 52 4. 米麴菌genomic DNA之萃取 53 5. 轉形株之檢定 54 5.1螢光顯微鏡 54 5.1.1 玻片培養之觀察 54 5.1.2 平板培養基培養之觀察 54 5.2螢光分析儀 54 5.3南方氏雜合分析 55 5.3.1 限制酶截切與DNA電泳 55 5.3.2 半乾式轉印 55 5.3.3 探針製備 55 5.3.4 雜合反應 56 5.3.5 呈色反應 56 5.4 西方式雜合分析 57 5.4.1 米麴菌粗胞內蛋白質萃取液之製備 57 5.4.2 蛋白質轉印 57 5.4.3 雜合反應 58 6. 轉形株之轉入基因穩定度測試 59 7. 重組蛋白質GMI之表達與免疫活性分析 59 7.1 重組蛋白質GMI之表達 59 7.1.1 液態培養(Liquid culture, LC) 59 7.1.2 固態基質培養(Solid-state culture, SC) 59 7.2 重組蛋白質GMI之純化 60 8. 重組蛋白質GMI之免疫活性分析 60 第三章、結果 61 1.質體建構 61 1.1 pPGFT/pPGMT 61 1.2 pPGAFT/pPGAMT 63 2. 米麴菌之pyrithiamine耐受度測試 65 3. 電穿孔轉形 65 4. 轉形株之檢測 66 4.1帶有報導基因gfp之轉形株 66 4.1.1 螢光顯微鏡觀察 66 4.1.2 南方氏雜合分析 70 4.1.3 西方氏雜合分析 71 4.1.4 螢光分析儀之檢測 74 4.2 帶有目標基因gmi之轉形株 75 4.2.1 西方氏雜合分析 75 4.2.2 南方氏雜合分析 79 5. 轉形株之轉入基因穩定度測試 80 6. 重組蛋白質GMI之表達與純化 80 7. 重組蛋白質GMI之免疫活性分析 82 第四章、討論 83 第五章、總結 91 第六章、未來展望 92 參考文獻 93 | |
| dc.language.iso | zh-TW | |
| dc.title | 以米麴菌表達由小孢子靈芝選殖之免疫調節蛋白質GMI | zh_TW |
| dc.title | Expression of the immunomodulatoryprotein, GMI, from Ganoderma microsporum in Aspergillus oryzae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許先業(Hsien-Yeh Hsu),常怡雍(Yee-Yung Charng),常玉強(Yuh-Chyang Charng),許瑞祥(Ruey-Shyang Hseu) | |
| dc.subject.keyword | 米麴菌,表達系統,基因融合,GMI,免疫調節蛋白質,電穿孔轉形法, | zh_TW |
| dc.subject.keyword | Aspergillus oryzae,expression system,gene fusion,GMI,immunomodulatory protein,electroporation, | en |
| dc.relation.page | 103 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-23 | |
| 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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