請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23856完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Chia-Chang Chung | en |
| dc.contributor.author | 張家禎 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:11:46Z | - |
| dc.date.copyright | 2006-08-09 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | 1. 1998. Green Fluorescent Protein: Properties, Applications, and Protocols.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23856 | - |
| dc.description.abstract | 塵螨是室內環境中常見的過敏原，其中源自於塵螨消化道上皮細胞的第二群過敏蛋白Dermatophagoides pteronyssinus 2（Der p 2）為導致氣喘等過敏症狀的過敏蛋白之ㄧ。台灣氣喘人口中有近百分之八十五對Der p 2過敏，其cDNA序列長度為387 bp，蛋白質由129個胺基酸組成，分子量約14 kD。
利用口服給予抗原蛋白，活化調節性T淋巴細胞，以降低抗原專一性的免疫反應，稱為口服耐受性（oral tolerance），此療法雖然便利有效，但卻受限於複雜的純化過程及昂貴之抗原蛋白。本研究嘗試以金針菇表現屋塵螨蛋白Der p 2，希望利用栽培技術成熟、方便安全可食用及低生態衝擊等優勢之菇類表達系統，開發口服減敏療法之新領域。 本研究分別以增加異源基因之5’端內含子、表現綠色螢光蛋白與der p 2融合基因、內質網停留訊號及慣用密碼子的更改等分生策略，建構表現質體，搭配電穿孔轉形方法進行金針菇菌絲體異源蛋白質之表達。 西方雜合分析顯示，以der p 2 cDNA所建構之表現質體於轉形後皆無明顯之Der p 2雜合訊號，未表現之轉形株以反轉錄-聚合酶連鎖反應確定mRNA正確存在，推測可能是異源基因轉譯層次的問題，進而根據金針菇最適密碼子變更der p 2為mder p 2，以mder p 2所建構表現質體之轉形株可由西方雜合分析可篩選到具Der p 2雜合訊號之菌株。接著由轉形株染色體DNA聚合酶連鎖反應及南方氏雜合分析，確定質體片段可經電穿孔轉形法插入轉形株染色體DNA中，具一至多個基因拷貝數。表現Der p 2之轉形株經三明治酵素免疫分析法（sandwich-ELISA）定量，其中轉形株pAImDS-19測得最高Der p 2表現量達可溶性蛋白之0.02%。 宿主的慣用密碼子或異源基因之G+C比例為金針菇表現系統中一相對重要之因子。在本研究中，利用金針菇表現屋塵螨蛋白Der p 2，不僅將菇類表現系統之蛋白質應用範疇向前推進，亦開創抗塵螨過敏食用疫苗之新型表現系統。 | zh_TW |
| dc.description.abstract | House dust mites are the most significant indoor allergens. Dermatophagoides pteronyssinus group 2 protein (Der p 2), originated from the digestive tract of host dust mites, plays a major role in induction of asthma, allergic rhinitis and allergic dermatitis. Up to 85% of asthma patients are sensitized to Der p 2 in Taiwan. There are 387 bp in the cDNA clone which encodes a 129-aa protein with a molecular mass of 14 kDa.
Oral tolerance can induce a group of antigen-specific regulatory T cells to suppress antigen-specific immune response through the oral administration of protein antigens presented by intestinal lymphoid tissue. However, the application of oral tolerance for the treatment of allergey was limited due to complicated purification and high cost of candidate allergens. To solve this problem, this study would like to develop the Der p 2-transgenic mushrooms possessing advantages of conveniently oral delivery and inexpensive protein production process. In this study, four different strategies were used to improve the heterogenous gene expression in Flammulina velutipes. Plasmids with an intron at 5’ of der p 2, the endoplasmic reticulum retention signal HDEL at 3’ of der p 2, fusion gene of der p 2 and egfp (enhanced green fluorescent protein), and modified the codon of der p 2 (mder p 2) were constructed for F. velutipes. After transforming the target DNA to mycelium by eleporation, only transformants of mder p 2 with an obvious signal were observed in Western blot analysis. Using Der p 2 sandwich enzyme-linked immunosorbent assay (sandwich-ELISA), the transformant pAImDS-19 expressed the highest Der p 2 protein, c.a. 0.02% of the total soluble proteins. This study demonstrated the expression of mite allergen in mushroom and suggested that the codon usage or the G + C content are crucial for the expression of heterogenous protein in F. velutipes. | en |
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| dc.description.tableofcontents | 目 錄 I
表目錄 V 圖目錄 VI 摘 要 VII Abstract VIII 第一章、緒言 1 一、過敏性疾病及免疫機轉 1 1. 人體免疫系統之平衡與調控 1 2. 過敏性疾病及衛生假說 2 3. 減敏療法與口服耐受性 3 二、塵螨與其過敏原蛋白 4 1. 家塵螨之相關研究 4 2. Der p 2的生化特性 5 3. Der p 2之異源表達與應用 5 三、真核微生物異源表達系統 9 1. 酵母菌 9 2. 絲狀真菌 9 3. 菇類表達系統現況 10 四、金針菇表達系統 13 1. 金針菇之相關研究 13 1.1 金針菇分類與形態特性 13 1.2 金針菇的栽培與利用 15 1.3 金針菇的藥用生理活性 15 2. 基因轉殖金針菇轉形系統 16 2.1表現型載體系統 16 2.1.1 啟動子 16 2.1.2 篩選標記 19 2.2 菇類異源表達之分生策略 21 2.2.1 內含子 21 2.2.2 內質網停留訊號胜肽 21 2.2.3 報導基因與目標基因融合策略 22 2.2.4 適用密碼子之選擇 22 2.3 轉形方法與寄主細胞之選擇 25 五、開發口服式減敏蛋白Der p 2基因轉殖金針菇之評估 26 六、研究目的 28 第二章、材料與方法 31 一、實驗材料 31 1. 菌株、質體及引子 31 二、實驗方法 38 I. 金針菇轉形系統 38 1. 質體建構 38 1.1 pAG及pAIG 40 1.2 pFD 40 1.3 pAID、pAIDH及pAIDS 40 1.4 pAIDGH及pAIDGS 40 1.5 pAIGD及pAIGDS 40 1.6 pAmD、pAImD、 pAImDS及pAImDGS 41 1.7 pAIMH及pAIMS 41 2. 質體轉形 49 2.1 質體DNA製備 49 2.2 勝任細胞製備 49 2.2.1 孢子法 49 2.2.2 菌絲法 49 2.2.3 細胞酵素處理 49 2.3 電穿孔轉形法 50 3. 轉形株檢定 50 3.1 DNA分析 50 3.1.1 金針菇染色體 DNA之抽取 50 3.1.2 聚合酶鏈鎖反應分析（Polymerase chain reaction） 51 3.1.3 南方氏雜合分析（Southern blot analysis） 51 3.2 RNA 分析 53 3.2.1金針菇RNA之抽取 53 3.2.3 限制酶截切分析與定序 54 3.3 重組蛋白質表現分析 55 3.3.1 螢光顯微鏡觀察 55 3.3.2 粗蛋白質萃取液之製備 55 3.3.3 點漬法分析（dot-blot） 55 3.3.4 西方雜合分析（Western blot analysis） 56 3.3.5 三明治法酵素親和吸附免疫分析（sandwich -ELISA） 56 4. 轉形株形態觀察 57 4.1 菌絲形態及細胞核數目觀察 57 II. Pichia pastoris轉形系統 58 1. 質體建構 58 1.1 pPD 58 1.2 pPDGH 58 1.3 pPGDH 58 2. 質體轉形 62 2.1質體DNA製備 62 2.2 勝任細胞製備 62 2.3 電穿孔轉形法 62 2.4 多插入基因數目轉形株篩選 63 3. 重組蛋白之表現分析 63 3.1 轉形株重組蛋白誘導表現 63 3.2 螢光顯微鏡觀察 63 3.3 蛋白質粗萃取液製備 64 3.4 西方雜合分析（Western blot analysis） 64 3.5 多株抗體製備 64 第三章、實驗結果 65 一、金針菇表現系統 65 1. 質體建構 65 2. 轉形結果與轉形株培養 70 3. 轉形株檢定 73 3.1 轉形株蛋白質表現分析 73 3.1.1 螢光顯微鏡觀察結果 73 3.1.2 點漬法分析（dot-blot） 73 3.1.3 西方雜合分析（Western blot analysis） 73 3.1.4 三明治酵素親和吸附免疫分析（sandwich -ELISA） 76 3.2 轉形株DNA分析 78 3.2.1 聚合酶連鎖反應與限制酶截切分析 78 3.2.2 南方氏雜合分析（Southern blot analysis） 80 3.3 轉形株RNA分析 82 3.3.1 反轉錄-聚合酶連鎖反應 82 3.4 轉形株形態分析 85 3.4.1 轉形株菌絲體染色顯微鏡觀察 85 3.4.2 轉形株菌絲體生長形態觀察 85 二、Pichia pastoris表現系統 87 1. 質體建構 87 2. Der p 2重組蛋白之表現 88 3. der p 2與egfp融合基因之表現 89 3.1 螢光顯微鏡觀察結果 89 3.2 西方雜合分析（Western blot analysis） 89 第四章、討論 92 一、金針菇異源表達系統之探討 92 1. 於金針菇中表現Der p 2之質體建構策略 92 1.1 5’端內含子 92 1.2 egfp與der p 2融合基因之表現 93 1.3 慣用密碼子 93 1.4 內質網停留訊號 94 2. 金針菇轉形效率與異源表現 94 二、der p 2於原核及真核系統表現之差異 95 第五章、結論 97 第六章、未來工作 98 第七章、參考文獻 99 | |
| dc.language.iso | zh-TW | |
| dc.title | 屋塵螨過敏原蛋白Der p 2於金針菇之異源表現 | zh_TW |
| dc.title | Study on expression of the house dust mite allergen Der p 2 in Flammulina velutipes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 江伯倫(Bor-Luen Chiang),常怡雍(Yee-yung Charng),李昆達(Kung-Ta Lee),許瑞祥(Ruey-Shyang Hseu) | |
| dc.subject.keyword | 金針菇,Der p 2,慣用密碼子,內質網停留訊號, | zh_TW |
| dc.subject.keyword | Flammulina velutipes,Der p 2,codon usage,endoplasmic reticulum retention signal, | en |
| dc.relation.page | 105 | |
| 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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