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博碩士論文 etd-0811117-060846 詳細資訊
Title page for etd-0811117-060846
論文名稱
Title
利用超高效液相層析質譜儀快速偵測及分型諾羅病毒外殼蛋白
Using M-class Ultra-Pure Liquid Chromatography/ Mass Spectrometry (UPLC/MSE) for Rapid Detection and Typing of Norovirus Capsid Protein
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
69
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2017-07-27
繳交日期
Date of Submission
2017-09-11
關鍵字
Keywords
基質輔助雷射脫附游離、類病毒顆粒、諾羅病毒、超高效液相層析儀、質譜儀
M-class ultra-performance liquid chromatography, mass spectrometry, matrix-assisted laser desorption ionization, Norovirus, virus-like particle
統計
Statistics
本論文已被瀏覽 5653 次,被下載 41
The thesis/dissertation has been browsed 5653 times, has been downloaded 41 times.
中文摘要
諾羅病毒(Norovirus)是非細菌性腸胃炎的主要致病源,統計顯示全球每年與諾羅病毒感染相關的有212,000件死亡案例。諾羅病毒是不具外套膜的單股RNA病毒,屬於杯狀病毒科 ( Caliciviridae )的一員。由三個開放讀碼框 (open reading frames;ORF)組成其RNA基因組結構,其中的ORF2負責決定諾羅病毒主要抗原性的外殼蛋白VP1之編碼。另外,依據VP1基因組序列,諾羅病毒分為GI到 GVII等七個基因群。諾羅病毒的VP1基因片段包含的殼結構域 (shell domain;S)突出結構域(protruding domain;P),突出結構域依序可進一步細分為P1第一次結構域 (the P1 sub-domain 1 )、P2次結構域 ( the P2 sub-domain )和P1第二次結構域 ( the P1 sub-domain 2 )。目前臨床實驗室針對諾羅病毒感染的檢測分析皆仰賴分子診斷。舉例來說,直腸拭子,嘔吐物或全糞便樣品皆透過反轉錄聚合酶鏈鎖反應(RT-PCR),即時定量PCR(RT-qPCR)及利用免疫層析法的快篩等技術分析。諾羅病毒具有高度的基因及抗原差異性。RT-qPCR的缺點是在無症狀的情況下容易得到偽陽性的結果,並缺乏定量數據成為了進一步臨床應用於腸胃道疾病的限制因素。透過基質輔助雷射脫附游離(matrix-assisted laser desorption ionization;MALDI)質譜技術直接偵測糞便提取物中的諾羅病毒外殼蛋白之偵測的方法也被提出。然而為了瞭解諾羅病毒之毒力、轉譯後修飾的可能性以及感染後的宿主細胞,本研究透過使用M級超高效液相層析儀(M-class ultra-performance liquid chromatography;M-class UPLC)和時間飛行Xevo®G2四極質譜儀(quadrupole time-of-flight mass spectrometer;Q-TOF / MS)將開發諾羅病毒感染快速定量鑑定,並進行基因分型及預測毒性反應的方法。
關鍵字:諾羅病毒;類病毒顆粒;基質輔助雷射脫附游離;超高效液相層析儀;質譜儀。
Abstract
Norovirus is the leading etiologic pathogen of diarrheal disease worldwide. By meta-analysis, norovirus was associated with 212,000 deaths annually, the disease burden of norovirus is high. Noroviruses are non-enveloped, single-stranded RNA viruses classified as genus Norovirus of the family Caliciviridae. The norovirus capsid is composed by VP1 proteins with shell (S) and protruding (P1 and P2) domains, in the order of S-P1-P2-P1 from N to C-terminus. The most variable P2 subdomain is insertion within the P1 domain. Based on the VP1 sequence, norovirus were divided into genogroups GI–GVII. GI, GII, and GIV are known to be the human pathogens, with GII being the most common in massive outbreaks. Due to norovirus are uncultivable in clinical laboratory, the combined reverse transcription polymerase chain reaction (RT- PCR) methods directly detect norovirus from specimens are widely used. However, the highly sensitive amplification might lead to false-positive results, while the high mutation rates of RNA viruses might lead to a negative result in the sequence dependent amplification. A GII.4 2006b norovirus like particle (VLP) was used to analysis for the signature signal of peptide profile. The matrix-assisted laser desorption ionization (MALDI) MS has used for examine the molecular weight of VLP. Total proteins from VLP were digested with trypsin, and separated on an M-class ultra-performance liquid chromatography (UPLC). Parallel ion fragmentation was programmed and signals were collected from 50 to 1500 m/z. Data was processed with ProgenesisTM QI-P for identification and quantification. Thus, the results in this study reveal that combination with more sensitive and rapid method for M-class UPLC and Xevo® G2 Q-TOF / MS has the potential to replace current detection methods. Keywords: Norovirus; virus-like particle; M-class ultra-performance liquid chromatography; mass spectrometry; matrix-assisted laser desorption ionization
目次 Table of Contents
目次
論文審定書………………………………………………………………………………i
誌謝 ……………………………………………………………………………………ii
中文摘要 ……………………………………………………………………………...iii
英文摘要 ……………………………………………………………………………...iv
第一章 前言 ……………………………………………………………………………...1
1. 諾羅病毒 ……………………………………………………………………………...1
1.1緣起 ……………………………………………………………………………...1
1.2 結構型態及性質 ……………………………………………………………………………...2
1.3臨床症狀及傳染途徑 ……………………………………………………………………………...4
1.4診斷 ……………………………………………………………………………...6
1.5 類病毒顆粒 (VIRUS LIKE PARTICLES, VLPS) ……………………………………………………………………………...8
2 質譜技術 ( MASS SPECTROMETRY,MS ) ……………………………………………………………………………...10
2.1基質輔助雷射脫附游離暨飛行時間串聯質譜儀 (MALDI-TOF MS) ……………………………………………………………………………...11
2.2 超高效液相層析質譜儀(M-CLASS ULTRA-PURE LIQUID CHROMATOGRAPHY, UPLC) 和時間飛行XEVO®G2四極質譜儀(QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER,Q-TOF/ MSE) ……………………………………………………………………………...13
3.研究動機 ……………………………………………………………………………...21
第二章 材料與方法 (圖一) ……………………………………………………………………………...23
1. 建立諾羅病毒VP1蛋白的類病毒顆粒表現系統 ……………………………………………………………………………...23
1.1 諾羅病毒之基因選殖 ……………………………………………………………………………...23
1.2 西方點墨法檢測 VP1 蛋白 ……………………………………………………………………………...23
1.3 電子顯微鏡觀察諾羅類病毒顆粒之型態 ……………………………………………………………………………...24
2. 基質輔助雷射脫附游離串聯飛行時間質譜儀分析……………………………………………………………………………...24
2.1 樣品準備與上機 ……………………………………………………………………………...24
3.超高效液相層析儀(M-CLASS ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY,M-CLASS UPLC)和時間飛行XEVO®G2四極質譜儀(QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER,Q-TOF / MSE)分析……………………………………………………………………………...25
3.1 層析管柱的參數設定 ……………………………………………………………………………...25
3.2 諾羅類病毒顆粒樣品備製 ……………………………………………………………………………...25
3.3 上機 ……………………………………………………………………………...26
3.4 數據分析 ……………………………………………………………………………...27
第三章 結果 ……………………………………………………………………………...28
1. MALDI-TOF MS測得諾羅類病毒顆粒殼蛋白質量 ……………………………………………………………………………...28
2 超高效液相層析儀串聯四極柱飛行時間質譜儀(UPLC/MSE )分析諾羅類病毒顆粒殼蛋白的表現 ……………………………………………………………………………...29
第四章 討論 ……………………………………………………………………………...31
參考文獻 ……………………………………………………………………………...37
附錄 ……………………………………………………………………………...58

圖次
圖一、實驗流程 ……………………………………………………………………………...50
圖二、諾羅類病毒顆粒以 MALDI-TOF 分析 ……………………………………………………………………………...51
圖三、PLGS 分析後序列覆蓋圖譜(COVERAGE MAP) ……………………………………………………………………………...52
圖四、分析M-CLASS UPLC/MSE的平行離子片段訊號 ……………………………………………………………………………...53
圖五、以 M-CLASS UPLC/MSE 分析後之離子片段訊號 (S DOMAIN) ……………………………………………………………………………...54
圖六、以 M-CLASS UPLC/MSE 分析後之離子片段訊號 (P1-SUB-DOMAIN) ……………………………………………………………………………...55
圖七、以 M-CLASS UPLC/MSE 分析後之離子片段訊號 (P2-SUB-DOMAIN) ……………………………………………………………………………...56
圖八、諾羅類病毒毒力代表片段以 M-CLASS UPLC/MSE 之離子訊號 ……………………………………………………………………………...57


附錄次
附錄圖一、諾羅病毒依據 RDRPOL 及VP1基因組序列的基因分群 ……………………………………………………………………………...58
附錄圖二、3D立體圖代表同樣的樣品在HPLC 及 UPLC 的表現上 ……………………………………………………………………………...59
附錄圖三、UPLC 及 HPLC於速度、解析度及敏感度的差異 ……………………………………………………………………………...60
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