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Title: | 利用酵母菌生產腸病毒七十一型類病毒顆粒 Production of enterovirus-71 virus-like particles in yeast |
Authors: | Tsung-Kai Huang 黃宗凱 |
Advisor: | 陳俊任(Chun-Jen Chen) |
Keyword: | 腸病毒七十一型,類病毒顆粒,嗜甲醇酵母菌,啤酒酵母菌, EV-71,VLPs,P. pastoris,S. cerevisiae, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | 腸病毒七十一型感染嬰幼兒後,會造成手足口症,甚至導致中樞神經系統癱瘓及死亡。早在 1998 年,就在臺灣爆發首次的大流行,但至今卻沒有疫苗供給預防。在此篇研究中,我們嘗試在嗜甲醇酵母菌及啤酒酵母中生產腸病毒七十一型類病毒顆粒作為疫苗的主要成份。在嗜甲醇酵母菌的表現系統中,所轉殖的基因位於 AOX1 promoter 下游,因此當以甲醇進行誘導時,基因得以表現。相對的,在啤酒酵母的表現系統中,轉基因則是持續地表現,並且在銅離子誘導後可使表現量提昇。共轉形結構性蛋白 (P1) 及病毒蛋白酶 (3C/3CD) 於此兩種系統後,產生了 VP0、VP1 及 VP3,意謂 P1 可以在酵母菌中成功地被 3C/3CD 截切。在破細胞後,以不連續式之蔗糖梯度離心以及免疫沉澱法純化,並以免疫金進行標定,類病毒顆粒可以在穿透式電子顯微鏡下被觀察到,此實驗所生產之類病毒顆粒將可合併之後生產的cholera toxin B (CTB),更進一步於動物實驗上進行免疫評估。 Enterovirus-71 (EV-71) infection causes hand, foot and mouth disease (HFMD) in children, and in some cases, it may also cause severe central nerve system collapse and even death. Since 1998, EV-71 has become an emerging infectious agent in Taiwan; however, there is currently no vaccine available to prevent the infection. In this study, we attempt to produce EV-71 virus-like particles (VLPs) in two yeast expression systems, including Pichia pastoris and Saccharomyces cerevisiae, and use the VLPs as a vaccine candidate. In the P. pastoris expression system, transgenes are placed under the AOX1 promoter which is activated upon methanol induction. In contrast, transgenes are constitutively expressed in the S. cerevisiae expression system and protein expression is enhanced after Cu2+ added. Co-transformation of genes encoding the structural protein P1 and the viral protease 3C/3CD leads to the production of VP0, VP1 and VP3 in both P. pastoris and S. cerevisiae expression systems, indicating that P1 can be successfully processed by 3C or 3CD in yeast cells. After cell lysis, discontinuous sucrose gradient centrifugation, and affinity purification with antibodies, VLPs can be detected under transmission electron microscopy (TEM) by immunogold staining. The EV-71 VLPs generated in this study will be used as a vaccine antigen to immunize animals with CTB in future experiments. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43353 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科技學系 |
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ntu-100-1.pdf Restricted Access | 4.21 MB | Adobe PDF |
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