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研究生:彭嘉冠
研究生(外文):Peng, Jia-Guan
論文名稱:日本腦炎病毒前膜蛋白螺旋區影響類病毒顆粒產出之研究
論文名稱(外文):Characterization of the prM Helix Domain for Virus-like Particle Production of Japanese Encephalitis Virus
指導教授:吳夙欽
指導教授(外文):Wu, Suh-Chin
學位類別:博士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:96
中文關鍵詞:日本腦炎前膜蛋白螺旋區病毒組裝
外文關鍵詞:Japanese encephalitisprMhelix domainvirus assembly
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日本腦炎病毒前膜蛋白 (prM) 與套膜蛋白 (E) 之間密切的交互作用是驅動病毒顆粒組裝之首要步驟。其中,前膜蛋白之helix區域 (prM-H domain) 除了能協助病毒顆粒之組裝,在病毒顆粒通過trans-Golgi network中之酸性環境進行重組後,會與套膜蛋白第二結構域 (E domain II, EDII) 密切接近,並可能與EDII形成新的交互作用。本研究針對日本腦炎病毒CH2195LA株的prM-H domain進行研究,利用基因點突變技術,探討 prM-H domain 與EDII之交互作用、以及對於類病毒顆粒 (virus-like particle, VLP) 釋放之影響。結果發現prM-H區域中的穀胺酸125殘基 (Glutamate125, E125) 對於VLP 之釋放極為重要,且其側鏈所帶之負電扮演關鍵性的角色。隨後並證明prM-E125 和EDII上帶正電之離胺酸93 (E-K93) 與組胺酸246 (E-H246) 殘基之間存在交互作用,而此交互作用也可能影響VLP 釋放。本研究更進一步以雙重突變prM-E125K/E-K93E 與 prM-E125H/E-H246E的方式交換正負電性,結果發現雙重突變並不影響交互作用,而其VLP也能順利形成並釋放。經由胺基酸序列比對後,發現 prM-E125 和 E-H246 殘基在不同黃質病毒中具有高度保留性,而E-K93殘基所帶之正電也能在不同黃質病毒中保留。由這些結果證明prM-E125與E-K93或E-H246之間產生的交互作用很可能來自於正負電性之相互吸引,並能協助VLP的釋放。針對這些保留性的氨基酸殘基,設法阻止或抑制其交互作用之形成,很可能成為有效對抗黃質病毒感染的新策略。
Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (EDII) following rearrangement triggered by low pH. In this study, the involvement of the prM-H domain of the Japanese encephalitis virus in VLP production and interaction with EDII was investigated. The prM-H domain was characterized using amino acid substitution mutations to disrupt interaction between prM-H and EDII; we then analyzed the effects of these substitutions on VLP production. The results indicate that substitutions of negatively charged prM-E125 residues with non-charged and positively charged residues significantly impaired both VLP production and the binding of prM-H oligopeptides to EDI/II proteins. Further, after making alanine substitutions of positively charged E-K93 and E-H246 residues (known to interact with the prM-H domain) at EDII resulted in decreased VLP production. Exchanges of oppositely charged residue side chains at prM-E125K/E-K93E and prM-E125H/E-H246E did not impair prM-H oligopeptide binding to EDI/II proteins, but did result in partial VLP production recovery. The prM-E125 and E-H246 residues are conserved and that the positive charge of the E-K93 residue is preserved in different flavivirus groups. These findings suggest that the electrostatic attractions of prM-E125, E-K93 and E-H246 residues are vital to flavivirus VLP production and inhibiting these interactions is a potential strategy for blocking flavivirus infections.
中文摘要
ABSTRACT
AKNOWLEDGEMENT
ABBREVIATIONS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
1. INTRODUCTION
1.1 Overview of Japanese encephalitis virus
1.1.1 Epidemiology
1.1.2 Transmission
1.1.3 Structure of JEV
1.1.4 Life cycle of JEV
1.2 JEV particle assembly
1.3 prM-E interaction
1.4 Roles of prM-H domain
1.5 Research goals
2. MATERIALS AND METHODS
2.1 Cells and antibodies
2.2 Bioinformatic predictions
2.3 Generation of plasmids containing JEV prM and E genes
2.4 Recombinant protein expression and purification
2.5 SDS-PAGE and Western blotting
2.6 Immunoprecipitation
2.7 VLP Production and purification
2.8 Enzyme-linked immunosorbent assay (ELISA)
2.9 Visualization of VLP by transmission electromicroscopy
2.10 Statistical analysis
3. RESULTS
3.1 JEV prM-H domain residues face toward EDII
3.2 VLP production by prM-E125 mutations in HEK293 cells.
3.3 prM-H oligopeptides bind with recombinant EDI/II
3.4 Positively charged residues at EDII are critical to prM-H binding and VLP production
3.5 Positively charged E-K93 and E-H246 residues interact with prM-E125
3.6 Sequence alignment analyses of prM-E125, E-K93 and E-H246 in different flavivirus groups indicate conserved residues
4. DISCUSSION
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