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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 黃慶璨 | |
dc.contributor.author | Yin-Hsi Lin | en |
dc.contributor.author | 林映希 | zh_TW |
dc.date.accessioned | 2021-06-17T06:13:39Z | - |
dc.date.available | 2022-01-25 | |
dc.date.copyright | 2019-01-25 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-09-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71888 | - |
dc.description.abstract | 諾羅病毒外鞘P蛋白質具有三個表面環狀結構適合作為抗原嵌入位。嵌有外來抗原的P蛋白質組裝成P粒子後,可大幅提高外來抗原的免疫原性,是一個近年來被廣泛研究,極具潛力的疫苗平台。更重要的是P蛋白質可以利用大腸桿菌或嗜甲醇酵母菌Pichia pastoris進行大量生產奠定了產業化的基礎。然而,現今以兩端固定在P蛋白質上的方式呈現抗原,卻可能因外來抗原的結構特殊,或體積龐大而破壞P蛋白質本身的結構,進而阻礙P粒子的正常組裝,這可能就是現在以諾羅病毒做為多抗原呈現平台時,多以呈現小片段胜肽為主的原因。因此,本研究嘗試將P蛋白質由第2個環狀結構的胺基酸位置拆成兩段(分別為PN及PC),分別利用兩個甲醇誘導的啟動子各自表現後,利用SDS-PAGE及西方墨點法進行胞內或胞外蛋白質的生產確認,再搭配蛋白質原態電泳、Ni-NTA pull down及Ni-NTA純化及膠體過濾法證實拆開的P蛋白質(PN及PC)可以重新組裝成完整的P蛋白質,更可以進一步組裝成P粒子。將來,利用拆開的P蛋白質呈現外來抗原時,可藉由一端游離的形式攜帶抗原,降低抗原大小及種類對P蛋白質結構造成的壓力,使諾羅病毒P粒子多抗原呈現平台更具彈性及應用性。 | zh_TW |
dc.description.abstract | As a vaccine platform, norovirus P particle not only can present antigens in a highly repetitive manner to enhance the immunogenicity of the foreign antigen, but also have three surface loops for antigens insertion in a P protein. Moreover, P protein can be produced in many host systems including E.coli and Pichia pastoris. However, the most common way to present whole protein antigen with two terminus linked to P protein might disrupt the structure of P protein, and consequently lead to the difficulty of P particle assembly. This might be the cause of difficulties of presenting large antigen or antigen with spatially distant N and C termini.
In order to overcome this obstacle, P protein was split into two parts from surface loop 2, so that antigen was presented with only one terminus fused, to decrease the conformational stress on P protein. Here we showed that the split P proteins (PN and PC) were successfully produced by P. pastoris and can be scaled-up by a 5-L fermenter. Ni-NTA pull-down assay, native PAGE and gel filtration data together showed that PN and PC interact with each other and could form multimers. The establishment of this split P protein vaccine platform might improve the flexibility of presenting different antigens. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:13:39Z (GMT). No. of bitstreams: 1 ntu-107-R05B22011-1.pdf: 23247376 bytes, checksum: e66b6d7ac6f71b81dd8bb8b9a3601f46 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 謝誌 2
摘要 3 目錄 5 圖目錄 10 第一章、 前言 12 一、 類病毒顆粒疫苗 12 1、 類病毒顆粒疫苗的發展優勢 12 2、 類病毒顆粒作為抗原呈現平台 13 3、 類病毒顆粒作為抗原呈現平台面臨的困難與因應之道 13 二、 諾羅病毒 15 1、 諾羅病毒分子生物學特性及分類 15 2、 諾羅病毒作為抗原呈現平台 16 3、 以諾羅病毒P粒子作為抗原呈現平台之優勢 17 三、 異源蛋白質表達系統 19 1、 嗜甲醇酵母菌P. pastoris表達系統 19 四、 研究動機與目標 21 1、 研究動機 21 2、 研究策略 22 3、 研究目標 22 第二章、 材料與方法 24 一、 藥品與培養基 24 二、 菌株與培養條件 28 1、 細菌 28 2、 真菌 28 三、 表現載體之建構 29 1、 pPICZαA-NoVP 30 2、 pPICZαA-PN-His6與pPICZαA-PC-His6 30 3、 pPICZB-PN 30 4、 pPIC3.5K-His6-PC 31 四、 嗜甲醇酵母菌-重組蛋白質表現系統 35 1、 嗜甲醇酵母菌P. pastoris勝任細胞製備 35 2、 P. pastoris電穿孔轉形 35 3、 轉形株篩選 36 4、 轉形株目標基因拷貝數檢定 36 5、 嗜甲醇酵母菌之誘導 38 (1) 搖瓶誘導 38 (2) 醱酵槽誘導 38 五、 蛋白質分析 39 1. 胞內蛋白質萃取 39 2. SDS聚丙烯胺膠體電泳(SDS-PAGE) 40 3. 原態電泳(Native Gel) 40 4. 西方墨點法 41 5. Ni-NTA pull down 42 6. His Trap Ni-NTA agarose親和層析法 42 7. 蛋白質濃縮 43 8. 膠體過濾法Size exclusion chromatography (SEC) 43 9. 蛋白質定量 43 第三章、 結果 47 一、 以Pichia pastoris外泌系統表現Split P蛋白質 47 二、 以Pichia pastoris表現胞內Split P蛋白質 51 1、 諾羅病毒PN蛋白質表現菌株的篩選與誘導 51 2、 諾羅病毒PN與PC蛋白質共表現菌株的篩選與誘導 51 3、 諾羅病毒母菌株PN-b及共表現菌株NC3的拷貝數檢定 52 三、 PN及PC兩蛋白質之間的交互作用關係 60 1、 Ni-NTA pull-down實驗測試 60 2、 親和性管柱層析Ni-column之純化測試 60 四、 以5 L醱酵槽量產胞內之PN及PC 65 1、 以BMGY培養基進行量產 65 2、 以BSM培養基進行量產 65 3、 搖瓶、BMGY醱酵及BSM醱酵誘導之蛋白質產量比較 66 五、 PN及PC自我組裝之能力 70 1、 原態電泳分析 Split P protein 70 2、 膠體過濾法Gel filtration分析目標蛋白質形成之多聚體大小 70 第四章、 結果討論 76 一、 外泌型及內生性PN及PC蛋白質形成雙體化現象之差異 76 二、 PN蛋白質產量在母菌株與NC3菌株中的產量差異 76 三、 共表現菌株NC3中PN及PC之拷貝數 77 四、 拆開之P 蛋白質與全長之P蛋白質自我組裝能力差異 77 五、 拆開之P 蛋白質組裝能力及其應用性之探討 78 第五章、 結論 80 第六章、 未來展望 81 一、 嘗試利用不同的策略提升PN及PC的表現量 81 二、 估算PN及PC間的交互作力強弱 81 三、 以Split P protein平台呈現特殊抗原 82 四、 嘗試由其他表面環狀結構拆開 82 第七章、 參考文獻 84 | |
dc.language.iso | zh-TW | |
dc.title | 利用諾羅病毒拆開之P蛋白質自我組裝形成P顆粒呈現抗原 | zh_TW |
dc.title | Presenting Antigens in Norovirus P Particle Formed by Automatically Assembled Split P Protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林晉玄(Ching-Hsuan Lin),張世宗,陳浩仁 | |
dc.subject.keyword | 諾羅病毒,疫苗平台,嗜甲醇酵母菌,外鞘P蛋白質,抗原呈現, | zh_TW |
dc.subject.keyword | Norovirus,antigen,P particle,vaccine platform,Pichia pastoris, | en |
dc.relation.page | 85 | |
dc.identifier.doi | 10.6342/NTU201804136 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-09-27 | |
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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