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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張明富 | |
| dc.contributor.author | Ting-Chun Yu | en |
| dc.contributor.author | 游婷珺 | zh_TW |
| dc.date.accessioned | 2021-06-12T17:57:33Z | - |
| dc.date.available | 2008-02-20 | |
| dc.date.copyright | 2008-02-20 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-01-30 | |
| dc.identifier.citation | 參 考 文 獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27191 | - |
| dc.description.abstract | 嚴重急性呼吸道症候群 （severe acute respiratory syndrome, SARS) 在 2002 年末至 2003 年之間襲捲了數十個國家，致死率約 9.6%，嚴重急性呼吸道症候群相關之冠狀病毒（severe acute respiratory syndrome associated coronavirus, SARS-CoV）被證實與 SARS 引起之非典型肺炎有關。SARS-CoV 為一含有套膜之冠狀病毒，其基因體為一單股正向的RNA，長約 30,000 個核苷酸，具有 14 個 ORFs，主要包括四個結構性蛋白質，membrane (M)、 envelope (E)、 spike (S) 和 nucleocapsid (N) 以組成病毒顆粒，其中 spike 為一鑲嵌在膜上的蛋白質，具有和宿主細胞受體結合的功能，進而產生細胞融合的現象。本研究著重於 SARS-CoV 感染宿主時，除了目前所知的受體蛋白質ACE2 (angiotensin-converting enzyme2)、DC-SIGN (dendritic cell specific ICAM-3-grabbing nonintegrin) 和 L-SIGN (liver/lymph node-specific ICAM-3-grabbing nonintegrin; also called CD209L or DC-SIGNR) 以外，是否還有其他宿主細胞表面因子參與其中。
先前對於SARS-CoV的受體蛋白質研究，許多僅表現spike蛋白質來做實驗，而本實驗室利用較能模擬實際生理狀況的類病毒顆粒(virus-like particles, VLPs) 來作為主要實驗材料。分別含有SARS-CoV三種主要結構蛋白質E、M和S的重組桿狀病毒共同感染昆蟲細胞Sf9，此時細胞同時表現三種結構蛋白質並組裝成SARS-VLPs而釋出細胞到培養液中，從培養液中分離以及蔗糖梯度離心 (sucrose gradient centrifugation) 純化之後，利用穿透式電子顯微鏡 (transmission electron microscopy) 確認形成正確的病毒顆粒構形，利用此SARS-VLPs與具有SARS-CoV受體ACE2之細胞株共同培養，在十分鐘觀察到spike會和ACE2結合形成複合體，並且經由免疫沉澱以及質譜儀分析，得知有三個細胞蛋白質可能和病毒感染過程相關，其中包含二個細胞骨架蛋白質actin和vimentin，以及一個鈣離子依存性磷脂質結合蛋白質annexin A2。進一步的實驗發現 vimentin 蛋白質在和 SARS-VLPs 反應十分鐘，可以被 anti-ACE2抗體免疫沉澱，由共軛焦顯微鏡也觀察到 spike 及 vimentin 在細胞膜上有共位現象，證實 vimentin可能參與在SARS-CoV感染細胞時形成的 spike-ACE2 複合體當中。未來工作將會釐清vimentin在病毒感染過程中實際上扮演的角色，以及其調控機制。 | zh_TW |
| dc.description.abstract | Severe acute respiratory syndrome (SARS) which emerged in the winter of 2002 caused a mortality rate as high as 9.6%. SARS-coronavirus (SARS-CoV) is a causative agent of SARS. It is an enveloped, positive single-stranded RNA virus with an RNA genome about 30,000 nucleotides in length, which encodes 14 open reading frames. The virus particle consists of four major structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). The S protein initiates virus entry by binding to cell surface receptors followed by conformational changes that lead to membrane fusion. The specific aim of this study is to identify surface molecules of host cells involved in the process of SARS-CoV infection.
Virus-like particles (VLPs) consist of three major envelope proteins E, M and S that mimic the organization and conformation of native virus particles. VLPs can undergo infection, following cell attachment and membrane fusion. In order to generate SARS-VLPs, Sf9 cells were co-infected with recombinant baculoviruses that express E, M, and S proteins. Four days post-infection, cells were lysed and SARS-VLPs were isolated from culture medium and purified by sucrose gradient centrifugation. Electron microscopic and immunogold labeling analysis of negatively stained SARS-VLPs exhibited SARS-CoV-like structures. In this study, spike protein was found to form complexes with ACE2 when SARS-VLPs were co-incubated with Vero E6 cells for 10 min. Immunoprecipitation followed by LC-MS/MS analysis identified the association of actin, vimentin and annexin A2 with ACE2. The specific interaction between ACE2 and vimentin was further confirmed by immunoprecipitation and Western blot analysis. In addition, colocalization of the spike protein of SARS-CoV and vimentin on the cell membrane was demonstrated by confocal microscopic analysis. Taken together, these results indicate that vimentin may form complexes with spike and ACE2 during virus infection. The role of vimentin involved in virus entry needs to be further studied. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-12T17:57:33Z (GMT). No. of bitstreams: 1 ntu-97-R94442025-1.pdf: 1533190 bytes, checksum: ab43e0f5098d3d90a62e0a4dd5afd854 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目 次
中文摘要 …………………………………………………Ⅰ 英文摘要 …………………………………………………Ⅱ 縮寫表 ……………………………………………………Ⅲ 緒論 ……………………………………………………… 1 材料來源 …………………………………………………17 實驗方法 …………………………………………………22 實驗結果 …………………………………………………41 討論 ………………………………………………………46 圖表 ………………………………………………………50 參考文獻 …………………………………………………65 | |
| dc.language.iso | zh-TW | |
| dc.subject | 波形蛋白質 | zh_TW |
| dc.subject | 嚴重急性呼吸道症候群 | zh_TW |
| dc.subject | 受體 | zh_TW |
| dc.subject | 血管緊張素轉化酶 | zh_TW |
| dc.subject | receptor | en |
| dc.subject | ACE2 | en |
| dc.subject | vimentin | en |
| dc.subject | SARS | en |
| dc.title | 細胞骨架蛋白質參與血管緊張素轉化酶2依賴性嚴重急性呼吸道症候群冠狀病毒棘蛋白質之結合 | zh_TW |
| dc.title | Cytoskeletal proteins are involved in the ACE2-dependent binding of SARS-CoV spike protein | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李芳仁,詹迺立 | |
| dc.subject.keyword | 嚴重急性呼吸道症候群,波形蛋白質,受體,血管緊張素轉化酶,2, | zh_TW |
| dc.subject.keyword | SARS,vimentin,receptor,ACE2, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-01-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-97-1.pdf 未授權公開取用 | 1.5 MB | Adobe PDF |
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