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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Hao-Ye Lin | en |
| dc.contributor.author | 林浩業 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:25:16Z | - |
| dc.date.available | 2017-08-15 | |
| dc.date.copyright | 2012-08-15 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65103 | - |
| dc.description.abstract | 菇類分子農場(mushroom molecular pharming)係以菇類為生物反應器，應用於醫藥用蛋白質或其他特定蛋白質的生產，具備安全性高、製程簡單和成本低廉的優勢，特別是在口服疫苗的開發。先前已於金針菇建立穩定的農桿菌媒介轉形系統表現異源蛋白質。然而，其實際應用與發展仍然受限於細菌性抗生素篩選標記與低表現量兩大問題。因此，本研究以金針菇單一點突變琥珀酸脫氫酶(succinate dehydrogenase, Sdh)作為同源性篩選標記(homologous selectable marker)，建立非細菌性抗生素篩選系統，提升轉基因菇類的生物安全性。此外，發現刪除金針菇甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase, GPD)啟動子部分序列可明顯提升下游基因表現量。將其應用於功能性蛋白質B型肝炎表面抗原(hepatitis B surface antigen, HBsAg)之表現，產量可達329.08 ± 35.37 ng/g total soluble protein。希望藉由部分刪除之啟動子提升產量與同源性篩選，應用於金針菇B型肝炎口服疫苗之開發展，拓展菇類分子農場未來的應用。 | zh_TW |
| dc.description.abstract | The mushroom molecular pharming refers to the use of mushrooms as bioreactor to produce valuable and pharmaceutical proteins, especially oral vaccines. Previously, a reliable Agrobacterium-mediated transformation for Flammulina velutipes has been established. However, the application was limited due to bacterial antibiotic selectable marker and low yield in heterologous proteins. Until now, the selectable markers using antibiotic resistance genes raise public concerns of biosafety. In this study, we developed a homologous selectable strategy via a single point mutation of succinate dehygrogenase to get transformants against harmless agent, carboxin. Besides, the heterologous gene expression was greatly enhanced by partial deletion of the glyceraldehyde-3-phosphodehydrogenase (gpd) promoters. We also successfully expressed the hepatitis B virus surface antigen (HBsAg) by partial deletion of gpd promoter with a maximum expression of 329.08 ± 35.37 ng/g total soluble protein. We demonstrated that the homologous selectable marker and the enhancement of protein expression using partial deletion of gpd promoter provide useful strategies in development of oral vaccines in F. velutipes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:25:16Z (GMT). No. of bitstreams: 1 ntu-101-R99b22024-1.pdf: 18729180 bytes, checksum: 2c642b931d91611bb00a7aacb3d3b17f (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 謝誌 I
摘要 II AbstractIII 目錄 IV 圖目錄 IX 附圖目錄 XI 表目錄 XII 第一章 前言 1 一、 分子農場 1 1. 植物分子農場應用現況 1 2. 植物分子農場之優勢 2 3. 植物分子農場潛在的風險 2 二、 菇類分子農場 3 1. 菇類分子農場之優勢 3 三、 B型肝炎菇類口服疫苗 4 1. 黏膜免疫系統 4 2. 轉殖作物口服疫苗發展現況 4 3. B型肝炎口服疫苗發展性 5 四、 金針菇概述 7 1. 分類與形態 7 2. 栽培與市場價值 7 3. 營養價值與藥理活性 8 五、 菇類轉形系統 9 1. 轉形系統 9 2. 菇類電穿孔轉形法 9 3. 菇類農桿菌媒介轉形法 10 六、 篩選標記 11 1. 細菌性抗生素篩選系統 11 2. 無篩選標記系統 12 七、 金針菇單一點突變同源性琥珀酸脫氫酶基因篩選系統 14 1. 單一點突變同源性琥珀酸脫氫酶基因篩選系統 14 2. 單一點突變同源性琥珀酸脫氫酶基因篩選系統之優勢 15 八、 分生策略提升異源蛋白質表現量 16 1. 持續性表現之啟動子 16 2. 內含子 16 3. 訊息胜肽 17 4. 密碼子最適化 17 九、 研究動機與目標 18 第二章 材料與方法 20 一、 菌株與培養條件 20 1. 金針菇雙核菌絲 20 2. 大腸桿菌Escherichia coli DH5α 20 3. 農桿菌Agrobacterium tumefaciens LBA4404 20 二、 核酸引子序列 21 三、 金針菇琥珀酸脫氫酶啟動子選殖 23 1. Genome Walking 23 2. 金針菇染色體DNA萃取 23 3. 限制酶截切金針菇染色體DNA 23 4. Adaptors製備 23 5. Ligation 24 6. 聚合酶連鎖反應 24 四、 農桿菌媒介轉形質體建構 27 1. 潮黴素抗性農桿菌媒介轉形載體 27 2. 萎鏽靈抗性農桿菌媒介轉形載體 28 3. 農桿菌媒介轉形載體建構方法 32 五、 農桿菌電穿孔轉形 34 1. 農桿菌勝任細胞製備 34 2. 農桿菌電穿孔轉形 34 3. 農桿菌轉形株篩選 34 六、 金針菇農桿菌媒介轉形 35 1. 農桿菌培養與前處理 35 2. 農桿菌與真菌共培養 35 3. 選擇性培養基篩選 35 七、 金針菇轉形株篩選與分析 37 1. 選擇性培養基篩選與繼代 37 2. 轉形株螢光顯微鏡觀察 37 3. 轉形株核酸分析 37 4. 轉形株目標蛋白質分析 40 5. 西方墨點法分析 42 6. 南方氏雜合法分析 42 第三章 實驗結果 45 一、 金針菇單一點突變同源性琥珀酸脫氫酶基因篩選系統建立 45 1. 金針菇琥珀酸脫氫酶啟動子選殖 45 2. 完整金針菇琥珀酸脫氫酶選殖與定點突變 46 3. 農桿菌電穿孔轉形 46 4. 金針菇農桿菌媒介轉形 46 5. 轉形株DNA genomic PCR分析 47 二、 部分刪除金針菇甘油醛-3-磷酸脫氫酶啟動子對下游基因表現量之影響 55 1. 農桿菌電穿孔轉形 55 2. 金針菇農桿菌媒介轉形 55 3. 轉形株綠色螢光蛋白質基因分析 56 4. 轉形株綠色螢光蛋白質分析 56 三、 功能性基因B型肝炎表面抗原於金針菇表現量 67 1. 農桿菌電穿孔轉形 67 2. 金針菇農桿菌媒介轉形 67 3. 轉形株B型肝炎表面抗原基因DNA genomic PCR分析 68 4. 轉形株B型肝炎表面抗原蛋白質分析 68 第四章 討論 75 一、 金針菇單一點突變同源性琥珀酸脫氫酶基因篩選系統建立 75 1. 不同啟動子對於單一點突變同源性琥珀酸脫氫酶基因表現 75 2. 萎鏽靈抗性確認 76 二、 部分刪除金針菇甘油醛-3-磷酸脫氫酶啟動子對下游基因表現量之影響 77 1. 轉形株之差異 77 2. 目標基因插入數量與位置 77 3. 啟動子核酸序列分析 78 三、 功能性基因B型肝炎表面抗原於金針菇表現量 80 1. B型肝炎表面抗原mRNA轉錄情形 80 2. B型肝炎表面抗原轉譯後修飾作用 80 3. 蛋白質抽取方式與抗體辨識 81 第五章 結論 83 第六章 未來展望 84 第七章 參考文獻 85 第八章 附錄 90 | |
| dc.language.iso | zh-TW | |
| dc.title | 建立金針菇同源性篩選系統及B型肝炎口服疫苗之開發 | zh_TW |
| dc.title | Establishment of homologous selectable system and development of hepatitis B virus oral vaccine in Flammulina velutipes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許瑞祥(Ruey-Shyang Hseu),楊啟伸(Chii-Shen Yang),楊健志(Chien-Chih Yang),黃介辰(Chieh-Chen Huang) | |
| dc.subject.keyword | 菇類分子農場,金針菇,甘油醛-3-磷酸脫氫酶,同源性篩選標記,單一點突變琥珀酸脫氫酶,B型肝炎口服疫苗, | zh_TW |
| dc.subject.keyword | Mushroom molecular pharming,Flammulina velutipes,glyceraldehyde-3-phosphate dehydrogenase,homologous selectable marker,a single point mutation of succinate dehygrogenase,hepatitis B virus surface antigen oral vaccines, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-01 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-101-1.pdf 目前未授權公開取用 | 18.29 MB | Adobe PDF |
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