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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Yi-Chun Liu | en |
dc.contributor.author | 劉怡君 | zh_TW |
dc.date.accessioned | 2021-05-20T21:37:19Z | - |
dc.date.available | 2015-08-18 | |
dc.date.available | 2021-05-20T21:37:19Z | - |
dc.date.copyright | 2010-08-18 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-16 | |
dc.identifier.citation | Aoki FY, Sitar DS (1988) Clinical pharmacokinetics of amantadine hydrochloride. Clin Pharmacokinet 14: 35-51
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10535 | - |
dc.description.abstract | RNA 聚合酶複合體在禽流感病毒複製過程扮演重要角色,由 PA, PB1與 PB2三個次單元所構成,負責轉錄與複製病毒 RNA,關係到禽流感病毒是否能順利複製病毒子代,感染更多宿主細胞,並決定其遺傳訊息是否能完整傳遞。相較於病毒表面封套上較易突變的醣蛋白 HA 與 NA,RNA 聚合酶複合體在各種病毒亞型間較保守,且其功能乃啟動於病毒感染早期,使其成為良好的抗流感藥物標的。
本論文根據本土禽流感病毒株 A/Chicken/Taiwan/2838V/00 (H6N1) 序列,經結構及免疫性預測,設計可能的抗原決定基,合成短鏈胜肽作為抗原,嘗試製備 RNA聚合酶各次單元體 (PA, PB1及 PB2) 之單株抗體。經酵素連結免疫吸附法 (ELISA) 及免疫染色法分析,確認得到具良好專一性的 PA 及 PB2單株抗體,此外亦得到 PB1之傳統抗血清。分析這些抗體特性,以競爭型 ELISA 初步確認 PA 及 PB2單株抗體之抗原決定位,並以免疫染色法測定 PA 及 PB2 之抗體型別。 為了觀察 RNA 聚合酶各次單元體,於受感染細胞中位移過程,我們使用抗體標定受 H6N1 亞型禽流感病毒株 (A/Wild duck/Ilan/2904/1999) 感染的 MDCK 細胞,確認 PA 及 PB2 單株抗體及 PB1 抗體,均可辨識被感染細胞上的原態抗原。此外,利用免疫共沉澱分析,發現 PA 及 PB1 皆可辨認一個約 53 kDa 大小的蛋白質。另一方面,使用免疫染色法定位 RNA 聚合酶各次單元體於二維電泳圖譜位置,結果 PA 單株抗體可於對應分子量及等電點辨認專一蛋白質點。 本論文所產抗體除了能作為專一性的檢測與觀察工具,更可能進一步應用於基礎研究,協助了解流感病毒 RNA 聚合酶的活化機制。 | zh_TW |
dc.description.abstract | RNA polymerase is a protein complex composed of three subunits including PA, PB1 and PB2, which play essential roles for the replication of avian influenza virus genome. Compared with the glycoproteins on the viral envelope (haemagglutinin and neuraminidase), RNA polymerase complexe shows more conservative in the protein sequences among various virus subtypes. Moreover, the function of this enzyme complex is initiated in the early-stage of viral replication. This makes RNA polymerase an ideal target for the anti-influenza virus therapeutics.
In this study, we used synthetic peptides as antigen for the subunits of the avian influenza RNA polymerase complex (PA, PB1 and PB2) monoclonal anibodies preparation. The peptides were determined based on the structural analysis and the immunogenicity prediction from a local avian influenza virus A/Chicken/Taiwan/2838V/00 (H6N1) sequences. The result of enzyme linked immunosorbent assay (ELISA) and Western blot analysis confirmed that the specificity of the monoclonal antibodies against PA and PB2, as well as the conventional antisera against PB1. The epitopes of these two monoclonal antibodies (anti-PA and anti-PB2) were estimated by using competitive ELISA. The isotypes of the antibodies were determined by Western blots. To follow the cellular movement of the RNA polymerase complexes in a living cell, MDCK cells were infected with the H6N1 virus (A/wild duck/Ilan/2904/1999). Then the target proteins were detected by indirect immunofluorescence assays. The results of indirect immunofluorescence assay shows these antibodies can recognize antigen in native form in infected cells. By using immunoprecipitation, a 53 kDa protein was identified as interacted with PA and PB1. On the other hand,the position of PA on the two-dimensional map was allocated by Western blot. The antibodies produced in this work might be used as a specific probe for viral analysis in order to explore the activation mechanism in RNA replication. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T21:37:19Z (GMT). No. of bitstreams: 1 ntu-99-R97b47211-1.pdf: 6925617 bytes, checksum: 526e5f8ce167a6e2334811254f965f38 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要..................................................3
ABSTRACT..................................................4 第一章 緒論...............................................5 1.1 簡介禽流感病毒 ........................................5 1.1.1 禽流感病毒的歷史背景.............................5 1.1.2 流感病毒之分類...................................7 1.1.3 流感病毒的構造與形態.............................8 1.1.4 流感病毒的基因體................................10 1.1.5 流感病毒的蛋白質簡介............................11 1.1.6 流感病毒的複製週期..............................13 1.1.7 流感病毒的變異..................................17 1.2 RNA 聚合?三聚體的重要性.............................19 1.2.1 RNA 聚合?複合體之功能..........................19 1.2.2 RNA 聚合?複合體之生成與運輸....................19 1.2.3 RNA 聚合?複合體與宿主因子作用..................20 1.3 研究動機與目的 .......................................23 第二章 材料與方法........................................24 2.1 病毒樣本處理方法.....................................24 2.1.1 雞胚培養病毒增殖...................................24 2.1.2 病毒樣本濃縮與純化.................................25 2.1.3 蛋白質定量.........................................25 2.2二維膠體電泳..........................................26 2.2.1 樣本處理(脫鹽及溶解)..............................26 2.2.2 第一維等電點聚焦...................................26 2.2.3 第二維 SDS-PAGE 膠體電泳...........................28 2.3抗體製備相關實驗......................................29 2.3.1 動物免疫法.........................................29 2.3.2 單株抗體製備 .......................................29 2.3.3 單株抗體大量製備...................................32 2.3.4 免疫球蛋白純化.....................................33 2.4 免疫學方法...........................................34 2.4.1 酵素免疫分析法.....................................34 2.4.1.1 一般酵素免疫分析法...............................34 2.4.1.2 競爭型酵素免疫分析法.............................35 2.4.2 免疫共沉澱法 .......................................35 2.4.3 單株抗體型別分析法.................................36 2.5 以禽流感病毒感染MDCK細胞.............................38 2.6 蛋白質生物素標定.....................................39 2.7 共軛焦顯微鏡分析.....................................40 第三章 結果..............................................41 3.1 禽流感病毒 RNA 次單元聚合?蛋白 PA、PB1 及 PB2 之抗體製備.......................................................42 3.1.1 以複合的結構與免疫性指標預測並設計抗原決定位....42 3.1.2 傳統免疫抗血清製備 .............................50 3.1.3 單株抗體製備....................................51 3.1.4 單株抗體大量製備................................52 3.2 禽流感病毒 RNA 次單元聚合?蛋白 PA 及 PB2 單株抗體特性分析.......................................................60 3.2.1 以酵素連結免疫吸附法檢定抗原決定基位置.............60 3.2.2 以競爭型酵素連結免疫吸附法進一步檢定可能的抗原決定基位置.......................................................60 3.2.3 PA 單株抗體型別檢定................................61 3.3 利用抗體觀察 RNA 聚合?各次單元體於受病毒感染的 MDCK 細胞內位移的情形...........................................67 3.3.1 以免疫螢光染色法觀察 MDCK 細胞中 PB1 及 PB2 位移...67 3.3.2 以免疫螢光染色法觀察 MDCK 細胞中 PB2 及 PA 位移....68 3.3.3 本研究利用抗體標定觀察 MDCK 細胞中 RNA 聚合?各次單元體,但尚無法得知其新生各次單元體組裝並位移至細胞核的過程.68 3.3.4 病毒感染 MDCK 細胞 24 小時後各次單元體訊號明顯減弱69 3.4 以 PA、PB1 及 PB2 抗體與雞胚尿囊濃縮液進行免疫共沉澱75 3.4.1 PA 及 PB1 抗體與雞胚尿囊濃縮液中約 53 kDa 分子作用並沉澱.......................................................75 3.4.2 免疫共沉澱所得結果可能為蛋白質降解所致.............76 3.5 PA、PB1 單株抗體可於二維電泳圖譜標定出單一明顯的蛋白質點.......................................................80 第四章 討論..............................................82 4.1 以合成胜?作為抗原製備的單株抗體可辨識原態分子..82 4.2 本文所產 PA 及PB2 單株抗體的抗原決定基與目前已知功能區域的關係...................................................83 第五章 結論..............................................86 第六章 參考資料..........................................87 問答錄...................................................92 附錄.....................................................96 | |
dc.language.iso | zh-TW | |
dc.title | 利用禽流感病毒 RNA 聚合酶 PA 和 PB2 之單株抗體探討其細胞感染機制 | zh_TW |
dc.title | Exploring the cellular infection mechanism of avian influenza virus RNA polymerases PA and PB2 by monoclonal antibodies | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 王金和 | |
dc.contributor.oralexamcommittee | 張世宗(Shih-Chung Chang),常怡雍,陳翰民 | |
dc.subject.keyword | 禽流感病毒 (AIV),RNA 聚合酶,抗體, | zh_TW |
dc.subject.keyword | avian influenza virus (AIV),RNA polymerases,antibodies, | en |
dc.relation.page | 106 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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