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標題: | 利用最佳化啟動子提升農桿菌媒介之金針菇表現系統蛋白質產量 Enhancement of protein expression in Agrobacterium-mediated transformation system of Flammulina velutipes by optimized promoter sequence |
作者: | Ying-Tzu Lyu 呂映慈 |
指導教授: | 黃慶璨(Ching-Tsan Huang) |
關鍵字: | 甘油醛-3-磷酸脫氫酶,啟動子,農桿菌媒介轉形法,金針菇,異源表現,B型肝炎表面抗原, glycerol dehyde-3- phosphate dehydrogenase (GPD) promoter,Agrobacterium-mediated transformation,Flammulina velutipes,heterologous expression,Hepatitis B virus surface antigen (HBsAg), |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 隨著異源基因表現系統的多元發展,分子農場的概念近年來快速發展,廣泛的用於醫藥用蛋白質或是其他特用產品的生產。食用性的作物也可應用在口服疫苗 (edible vaccine) 的開發。其中的關鍵技術包括宿主的選擇、基因的表現策略,基因穩定性、表現量的提升以及免疫效力等。本研究選用市場普及可工業化生產的金針菇 (Flammulina velutipes) 為表現宿主,採用農桿菌媒介法轉入外來基因。前人的研究顯示,農桿菌媒介法可使基因穩定的嵌入染色體,不易因多次繼代而被剔除,但其發展性卻受限於低表現量,因此提升蛋白質產量為首要之務。因此在本研究中,比較不同長度的金針菇甘油醛-3-磷酸脫氫酶 (glycerol dehyde-3- phosphate dehydrogenase, GPD) 啟動子對於蛋白質表現量的影響。
結果顯示當gpd啟動子分別刪除114 bp和637 bp後,可得到表現量最高的轉形株,綠螢光蛋白質 (enhanced green fluorescent protein, eGFP) 的表現量於每克總可溶蛋白質中,最高可達4.38 毫克(mg);顯示當刪除特定片段後,可能去除啟動子上的轉錄因子結合位 (transcriptional factor binding sites),或是造成二級結構的改變,增加基因的轉錄效率,因此大幅提升表現量。本研究亦成功以全長gpd 啟動子於金針菇中表現B型肝炎病毒所產生蛋白質的表面抗原 (Hepatitis B virus surface antigen, HBsAg),但每克的總可溶蛋白質中,僅佔28.4奈克(ng),未來亦將採用刪除特定長度的gpd啟動子,提升HBsAg在金針菇的表現量。 Molecular farming has attracted extensive attention in the production of various pharmaceutical proteins. A lot of efforts have been put in developing edible vaccines by using transgenic plants to provide protection from infectious diseases. However, the success of using mushrooms as effective delivery vehicles to induce oral immunization is limited due to the difficulty in their transformation efficiency and gene expression. In comparison with conventional DNA-mediated transformation, Agrobacterium tumefaciens-mediated transformation (ATMT) appeared to be a more efficient tool which can be applied to kinds of fungal starting materials such as conidia, vegetative mycelia and fruiting body. The enoki mushroom Flammulina velutipes, which was chose as an expression host, is a popular edible mushroom in Asia. While the transformants remained stable, the low gene expression limited its practical application. In this study, I demonstrated that the heterologous gene expression can be greatly enhanced by appropriately trimming off the gpd promoters. As 114 bp and 637 bp was excised from the promoter, protein expression level would increase dramatically as much as 1000-fold. I also tried to express the human hepatitis B virus surface antigen (HBsAg) in F. velutipes. HBsAg was successfully expressed by using a full-length gpd promoter, showed the feasibility of using F. velutipes for edible vaccine development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45817 |
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顯示於系所單位: | 微生物學科所 |
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