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研究生:劉子瑄
研究生(外文):Tzu-Shuan Liu
論文名稱:表達諾羅病毒2006-2008台灣株之類病毒顆粒並探討其抗原性
論文名稱(外文):Expression and Characterization of Virus-like Particles of 2006-2008 Norovirus Strains in Taiwan
指導教授:黃智生黃智生引用關係
指導教授(外文):Jason C. Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:71
中文關鍵詞:諾羅病毒類病毒顆粒
外文關鍵詞:NorovirusVirus-like particle
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諾羅病毒(Norovirus ;NoV)是引起人類非細菌性急性腸胃炎的主要致病原。主要症狀為嘔吐及腹瀉,有時患者會引發嚴重的併發症狀像是脫水、抽搐甚至死亡。由於諾羅病毒目前尚未能在細胞株內繁殖培養,也無有致病症狀的動物模式可提供研究,所以開始使用重組蛋白系統表達出諾羅病毒的類病毒顆粒(Virus-like particle ;VLP),其具有保留原始病毒的抗原性且不帶有核酸所以不具有致病力的優點,因此成為研究諾羅病毒的重要工具。現今人類諾羅病毒分成兩個主要的基因群:GI和GII,且兩基因群又細分成許多的基因型。在全球群聚感染事件當中,諾羅病毒GII/4為主要引起流行的諾羅病毒基因型,而在台灣GII/4亦為最常由群聚感染檢體中檢測出的基因型。近期,根據台灣疾病管制局的長期監測發現,有兩株未經報導過的新型GII/4台灣本土突變病毒株出現。
  故在本篇研究中,我們利用Bac to Bac桿狀病毒系統表達2006-2008年間台灣本土新型的諾羅病毒類病毒顆粒,並利用電顯以及酵素結合免疫吸附分析法(ELISA)等技術比較類病毒顆粒與真實病毒的型態和抗原性。並進一步將產出的類病毒顆粒免疫BALB/c小鼠取得血清後檢測其對應不同型別病毒株之能力。結果顯示,使用新型病毒株類病毒顆粒免疫小鼠後的血清擁有較廣的交叉反應。另一方面,我們也利用大腸桿菌系統表達諾羅病毒外殼蛋白上變異性最高的P domain 區域,並利用類病毒顆粒免疫小鼠抗體去分析其抗原性,結果發現P domain蛋白形成的P粒子具有型別間專一性較高之特性。總結而言,使用新型的台灣本土病毒株的類病毒顆粒以及P粒子做為抗原免疫動物後所得的血清,未來可應用於改良和發展提升台灣病毒株敏感性及專一性較高的臨床檢測系統。

Noroviruses (NoVs ) are important human pathogens known to cause epidemic nonbacterial acute gastroenteritis or even life-threatening complications. Researches of Norovirus are limited by lacking a small-animal model and a reliable cell culture system. Recently, recombinant expression systems have been developed to express virus-like particles (VLPs) which presenting the antigen of the live virus while lacking the ability to reproduce or cause illness. Human Noroviruses are genetically divided into two major genogroups, GI and GII, each with a number of genotypes. Here in Taiwan, the genogroup II genotype 4 (GII4) is the predominant genotype. One study reported by Taiwan CDC has identified two new strains which were never been isolated in the past.
In this study, we expressed and characterized the virus-like particles of the newly isolated Taiwan Norovirus strains; after identified, these VLPs can be used for further studies. Using EM and ELISA methods, the VLPs expressed by bac-to-bac expression system in my study are determined to have similar morphology and antigenicity to live viruses. These VLPs were then used as the antigen to immunize BALB/c mice and the VLP-immunized mouse serum were collected for further study. Data from serology test have shown that these new strains have broad-range cross-reactivity among 2006-2008 strains. In order to clarify the specific reorganization of our VLP-immunized serum, we also expressed P domain (named as P particle), the highest variable region of capsid protein, by an E.coli expression system. The results suggest that VLP-immunized serum can specifically recognize P particles from homotypic strain but have cross-interaction between heterotypic VLPs. Taken together, expression of VLPs of new Norovirus strains which were used to produce VLPs-immunized serum are essential and the anti-sera can be potential components to set up a higher sensitivity antigen-capture ELISA improving the sensitivity and specificity of diagnostic rate in clinical infection.

論文電子檔著作權授權書……………………………………………………… i
論文審定同意書………………………………………………………………… ii
致謝……………………………………………………………………………. iii
中文摘要……………………………………………………………………… iv
英文摘要 ……………………………………………………………………… v
目錄………………………………………………………………………… vi
第一章 緒論…………………………………………………………………………1
第一節 諾羅病毒簡述………………………………………………………1
第二節 諾羅病毒類病毒顆粒的發展與應用…………………………………4
第三節 諾羅病毒與疾病………………………………………………………6
第四節 研究動機與目的…………………………………………………… 9
第二章 材料與方法…………………………………………………………………10
第一節 實驗材料……………………………………………………………10
第二節 實驗方法……………………………………………………………19
第三章 實驗結果 …………………………………………………………………34
第一節 諾羅病毒台灣株之類病毒顆粒表達與特性分析…………………34第二節 製備血清抗體與病毒型別間交叉反應之探討……………………35
第三節 諾羅病毒台灣株 P 粒子表達與特性分析…………………………36
第四節 類病毒顆粒與免疫血清在臨床檢測的應用………………………37
第四章 實驗討論……………………………………………………………………39
第五章 參考文獻……………………………………………………………………46
第六章 結果圖表……………………………………………………………………52
第七章 附錄…………………………………………………………………………67

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