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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳俊任 | |
| dc.contributor.author | Hui-Yi Wang | en |
| dc.contributor.author | 王惠儀 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:48:15Z | - |
| dc.date.available | 2017-08-28 | |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-13 | |
| dc.identifier.citation | 1. Bible, J. M., P. Pantelidis, P. K. Chan, and C. Y. Tong. 2007. Genetic evolution of enterovirus 71: epidemiological and pathological implications. Reviews in medical virology 17: 371-379.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64456 | - |
| dc.description.abstract | 腸病毒七十一型(enterovirus-71; EV-71)感染後，會造成手足口症，且易引起神經性併發症及死亡，在五歲以下的幼兒中有極高的致死率。於 1998 年時，在臺灣即爆發首次的大流行，然而目前卻沒有EV-71 的疫苗藥物供給預防。本研究之目的，在探討以嗜甲醇酵母菌表現系統生產的 EV-71 類病毒顆粒之最佳純化條件，並評估以其作為抗原之免疫效果。首先以一次非連續蔗糖梯度超高速離心純化 EV-71 類病毒顆粒，並將之進行動物免疫，然而成效不佳，推測可能肇因於類病毒顆粒之純度不高。因此進一步比較不同純化方法，包含蔗糖梯度與氯化銫梯度超高速離心，對類病毒顆粒之純度及產量的影響。在不同方式純化後，分別以SDS-PAGE與Western-blot分析類病毒顆粒之純度，並以穿透式電子顯微鏡觀察類病毒顆粒的產生。比較不同方法之純化結果，發現以二次非連續蔗糖梯度超高速離心純化方式，可得到最高的類病毒顆粒純度 (約 45%) 與總產量 (約 0.88 mg/L)。另外，在氯化銫梯度超高速離心後，得知EV-71類病毒顆粒的密度約為 1.22 g/ml。以二次非連續蔗糖梯度純化之類病毒顆粒作為抗原於小鼠進行免疫後，可有效引起體液免疫反應，其誘發之EV-71 專一性 IgG 抗體量，與以熱去活化 (heat-inactivated) EV-71為抗原時相當。綜合本研究之結果，顯示以酵母菌生產之EV-71類病毒顆粒具有發展成為疫苗之潛力。 | zh_TW |
| dc.description.abstract | Enterovirus-71 (EV-71) infection causes hand, foot and mouth disease (HFMD) and is prone to be complicated by severe neurological diseases, causing significant morbidity and mortality in children under 5 years of age. In 1998, EV-71 resulted in a large outbreak in Taiwan; however, there is no effective vaccine for EV-71 to date. In this study, we investigated the optimal method for purifying EV-71 virus-like particles (VLPs) expressed in Pichia pastoris, and evaluated the immunogenicity of purified VLPs in mice. We first purified EV-71 VLPs by a single discontinuous sucrose gradient ultracentrifugation, and found that the purified VLPs did not stimulate a strong immune response in mice, which was probably due to the presence of impurity in VLP samples. Therefore, we compared the purification of VLPs using different methods, including discontinuous sucrose gradient and CsCl gradient ultracentrifugations, and analyzed the purity and yield of VLPs by SDS-PAGE and Western blot. The results indicated that the highest VLP purity (about 45%) and yield (0.88 mg/L) could be obtained using double discontinuous sucrose gradient ultracentrifugation. The density of EV-71 VLP was determined to be about 1.22 g/ml by CsCl density gradient. Immunization of mice with the VLPs purified by double sucrose density gradient led to the production of anti-EV-71 IgG antibody, with a titer similar to that produced when heat-inactivated EV-71 was used as the immunogen. Taken together, our results indicate that the EV-71 VLPs produced in yeast have the potential to be developed as a vaccine candidate. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:48:15Z (GMT). No. of bitstreams: 1 ntu-101-R99b22047-1.pdf: 3188667 bytes, checksum: 67c85dc5c6259d20ac4e78ff85b13b8b (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 誌謝.......I
中文摘要..II Abstract..III 目錄......IV 圖表目錄..VIII 壹、前言........................................1 一、腸病毒七十一型 (enterovirus 71, EV-71)......1 1.1 源起........................................1 1.2 分類........................................1 1.3 病毒結構與生化特性..........................3 1.4 病毒生活史..................................4 1.5 病毒流行病學................................4 二、疫苗與類病毒顆粒............................5 2.1 疫苗........................................5 2.2疫苗作用原理及種類...........................5 2.3 類病毒顆粒 (virus like particles, VLPs).....6 2.4 腸病毒疫苗之研究............................8 三、嗜甲醇酵母菌表現系統........................8 3.1 簡介........................................8 3.2 甲醇代謝....................................9 3.3 酒精氧化酵素 (AOX)..........................9 3.4 菌株及表現載體..............................10 3.5 醣基化修飾..................................11 3.6 酵母菌生產之優勢............................11 四、病毒純化....................................12 4.1 簡介........................................12 4.2 區帶離心法..................................12 4.3等密度平衡離心法.............................12 貳、實驗目的與架構..............................14 參、材料與方法..................................15 一、微生物及載體................................15 1.1 病毒株......................................15 1.2 細胞株......................................15 1.3 菌株........................................15 1.4 載體........................................15 二、培養基配製..................................15 三、EV-71 類病毒顆粒之誘導表現及純化............16 3.1 蛋白質誘導..................................16 3.2 破菌........................................17 3.3 粗蛋白濃縮..................................17 3.4非連續蔗糖梯度離心...........................17 3.5 氯化銫密度梯度離心..........................18 3.6 非連續蔗糖梯度結合氯化銫密度梯度離心........ 18 3.7 二次非連續蔗糖梯度離心......................19 3.8 VLPs 濃縮與蛋白質定量.......................19 四、純化結果分析................................20 4.1 蛋白質電泳 (SDS-PAGE).......................20 4.2 西方點墨法 (Western blotting)...............20 4.3 銀染 (silver staining)......................20 五、穿透式電子顯微鏡 (Transmission Electron Microscopy, TEM)............................................21 5.1 TEM 觀察....................................21 5.2 免疫金標定 (immunogold labeling)............21 六、Ev-71 病毒培養與定量........................22 6.1 病毒增殖 (virus propagation)................22 6.2 病毒斑分析 (plaque assay)...................22 6.3 病毒生長曲線................................22 6.4 病毒大量培養及純化..........................23 七、EV-71 類病毒顆粒免疫活性評估................23 7.1 EV-71 VLPs免疫小鼠模式......................23 7.1.1 免疫小鼠模式 (一).........................23 7.1.2 免疫小鼠模式 (二).........................24 7.2 EV-71 專一性抗體分析........................24 7.3 脾臟細胞增生試驗............................25 7.4 細胞激素測定................................25 八、統計與繪圖軟體之分析........................25 肆、結果........................................26 一、EV-71 病毒培養與純化........................26 1.1 EV-71 生長曲線..............................26 1.2 EV-71 之純化................................26 二、EV-71 類病毒顆粒純化及免疫活性評 (一).......26 2.1 非連續蔗糖梯度離心..........................26 2.2 EV-71 專一性抗體分析........................27 三、EV-71 類病毒顆粒純化及免疫活性評估 (二).....27 3.1氯化銫密度梯度離心...........................27 3.2非連續蔗糖梯度離心結合氯化銫密度梯度離心..... 28 3.3 二次非連續蔗糖梯度離心......................29 3.4 EV-71 專一性抗體分析........................30 3.5 EV-71 特異性之脾臟細胞增生反應測定..........30 3.6 細胞激素測定.................................31 伍、討論 32 陸、結論 36 柒、參考文獻 37 捌、圖 48 玖、表 69 拾、附錄 70 | |
| dc.language.iso | zh-TW | |
| dc.subject | 腸病毒七十一型 | zh_TW |
| dc.subject | 類病毒顆粒 | zh_TW |
| dc.subject | 疫苗 | zh_TW |
| dc.subject | Key words：enterovirus-71 (EV-71) | en |
| dc.subject | virus-like particles (VLPs) | en |
| dc.subject | vaccine | en |
| dc.title | 腸病毒七十一型類顆粒之純化及其免疫活性之評估 | zh_TW |
| dc.title | Purification and immunogenicity of enterovirus-71 virus-like particles | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 龔思豪,朱清良 | |
| dc.subject.keyword | 腸病毒七十一型,類病毒顆粒,疫苗, | zh_TW |
| dc.subject.keyword | Key words：enterovirus-71 (EV-71),virus-like particles (VLPs),vaccine, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-14 | |
| 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 | |
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