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系統識別號 U0026-2808201601034800
論文名稱(中文) 低溫電子顯微鏡解析成熟登革類病毒的構型
論文名稱(英文) Cryo-EM reveals the conformation of mature dengue virus-like particle
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 陳聖壬
研究生(英文) Sheng-Ren Chen
學號 T46031038
學位類別 碩士
語文別 英文
論文頁數 44頁
口試委員 指導教授-吳尚蓉
召集委員-王淑鶯
口試委員-趙黛瑜
中文關鍵字 低溫冷凍電子顯微鏡  登革類病毒  單粒子分析  三維結構重組 
英文關鍵字 Cryo-electron microscopy  dengue virus-like particle  single particles analysis  three-dimensional reconstruction 
學科別分類
中文摘要 登革熱是由埃及斑蚊 (Aedes aegypti)與白蚊伊蚊 (Aedes albopictus)攜帶登革熱病毒所引發的疾病。登革熱所造成的病徵輕則高燒、頭痛、肌肉痛、關節痛;重則引發登革出血熱 (Dengue hemorrhagic fever, DHF)或登革休克熱 (Dengue shock syndrome, DSS),甚至導致死亡。登革熱病毒屬於黃病毒科 (Flaviviridae)的黃病毒 (Flavivirus)。從外觀來看,登革熱病毒屬於外套膜病毒,並且具有二十面體對稱性。登革熱病毒有四種血清型態。當感染某一型痊癒後,會對此型終生免疫,但反而增加了感染其他型的風險。登革疫苗發展至今,仍未發展出有效對抗登革熱病毒複製的疫苗或藥物。在2014年以及2015年夏天,分別在高雄與台南爆發了嚴重的登革熱疫情,造成大量因登革熱死亡的病例,因此研發有效的登革疫苗與藥物是燃眉之急的。
登革熱病毒由caspid protein (C)、enveloped glycoprotein(E)、precursor membrane/ membrane glycoprotein(prM/M)三種結構性蛋白所組成。登革熱病毒在成熟過程中會經歷一系列的結構變化。我們從中興大學趙黛瑜實驗室取得成熟登革類病毒顆粒(dengue virus-like particle, DENV VLP),它是從含有E及prM蛋白的質體轉染COS-1細胞所分泌出的類病毒顆粒。登革類病毒顆粒不具感染性,並證實可以誘發較高的抗體中和反應,因此是很好的候選疫苗之一。在這裡我們利用低溫電子顯微鏡與三維結構重組技術來解析成熟登革類病毒顆粒結構,從結構來了解類病毒如何保留抗原決定位並引發較高中和反應。結構上獲得的資訊將可提供未來製藥及疫苗開發的依據。
英文摘要 Infection with Dengue Virus (DENV) usually causes fever accompanied by rashes and joint pain in patients but might lead to dengue hemorrhagic fever (DHF) and dengue shock (DSS) syndrome, which may be fatal. The DENV is normally carried by Aedes aegypti mosquito and Aedes albopictus mosquitoes. DENV belongs to the flavivirus genus of the Flaviviridae family and has four serotypes. When primary infected with one of four serotypes, it will produce lifelong immunity effect for this serotype. But increase the risk of other DENV serotypes infection. So far, there is no effective vaccine or therapeutic drugs against DENV infection. Last two years (2014 and 2015), there were large-scale outbreak of dengue fever in Kaohsiung and Tainan that caused thousands of dead medical cases. Hence, providing information from structural point of view for future drug and effective dengue vaccine development is imperative.
Dengue virus is consisting of three structural proteins, the capsid (C), the envelope (E) and the premembrane/membrane (prM/M) proteins, that form the virus particle. The virus will undergo a series of conformational changes during the maturation process. We cooperated with Prof. Day-Yu Chao, National Chung Hsing University and obtained mature DENV virus-like particle (VLP). It was plasmid DNA directly synthesis of secreted prM and E proteins by the host cells, COS-1 cell. DENV VLP was highly immunogenic and noninfectious that had potential as biosynthetic subunit vaccines. Here, cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction approach were used to reveal the mature DENV VLP structure and told us how the neutralizing epitopes are preserved on the surface of VLPs. The information of the viral structure would be helpful for antiviral candidates and effective vaccine development.
論文目次 中文摘要 i
Abstract ii
Acknowledgement iii
Catalogues iv
Table List viii
Abbreviations ix
1. Introduction 1
1.1 Dengue virus 1
1.1.1 Dengue virus structure 2
1.1.2 The life cycle of DENV and its conformational change 3
1.1.3 Different DENV morphologies increase the difficulty and complexity for dengue vaccine development 4
1.2 Dengue virus-like particles 5
1.3 TEM technology 6
1.3.1 Cryo-EM (Cryo- Electron Microscopy) 7
1.4 Single-particles analysis 7
2.Rationale 9
3.Materials and methods 10
3.1 Mature dengue virus-like particles preparation 10
3.2 Transmission Electron Microscopy facility 10
3.3 Negative-stain EM 11
3.4 Immunogold labeling 11
3.5 Cryo-electron microscopy (Cryo-EM) images 12
3.6 Three-dimensional reconstruction of mature DENV VLP 13
4.Results 14
4.1 Identify the mature DENV VLPs 14
4.1.1 Negative stain-EM 14
4.1.2 Immunogold labeling 14
4.2Three-dimension reconstruction of mature DENV VLPs 15
4.2.1 Cryo-EM images 15
4.2.2 Analyze the heterogeneity of particles 16
4.2.3 3D structure of mature DENV VLPs 16
5.Discussion 18
6.Conclusion 24
7.References 25
8.Figures 32
9.Tables 43
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