臺灣博碩士論文加值系統

PB2是A型流行性感冒病毒的病毒蛋白質，研究認為PB2的第627個位點是造成宿主限制的重要因子，該位點胺基酸在禽流感病毒中多是Glutamic acid；而人流感病毒中則多是Lysine。分析從1918年至今，H1N1亞型禽流感和人流感的PB2胺基酸序列，針對已發表之在PB2上其他可區分禽流感和人流感的位點，發現199、475、627位點在演化上具有保留性。為了探討這三個位點可能扮演的角色，我們建立A/WSN/33 (H1N1) 的反轉基因學系統，在PB2上進行單點及多點的突變。單點突變之627E avian-like PB2會降低vRNP活性，而兩點突變之199A+475L avian-like PB2也會顯著降低vRNP活性。帶有avian-like標誌的PB2，不論是否表現viral-like RNA，627E PB2和NP的交互作用和wild type相比都較差；在表現viral-like RNA的狀態，帶有199A+475L avian-like突變的PB2和NP的交互作用小於wild type和627E avian-like PB2；但是在不存在viral-like RNA的狀態，帶有199A+475L avian-like突變的PB2和NP的交互作用大於wild type和627E avian-like PB2。由以上結果得知，PB2除了627位點以外，199和475位點在人類細胞中可能也是具有影響vRNP活性的位點，可以藉由不同功能影響PB2本身或PB2和其他蛋白質的作用。

PB2 is a viral protein of influenza A virus. The position 627 of PB2 is considered as a determinant of host range restriction. In general, avian influenza contains glutamic acid and human influenza contains lysine at the position. Other positions on PB2 which can distinguish avian and human strains were identified by full-genome sequence analysis. We analyzed both avian and human H1N1 PB2 sequences since 1918. Position 199, 475, and 627 on PB2 are conserved evolutionally as human-like signatures in human influenza. To understand the potential roles of these 3 positions on vRNP functions, reverse genetics system based on the backbone of human influenza A/WSN/33 (H1N1) was used to address the mechanism. Mutant PB2 by single or multiple mutations on the 3 positions were generated. The double avian-like mutant, 199A+475L PB2 reduced RNP activity obviously as the single avian-like mutant, 627E PB2. Immunoprecipitation results showed that the bonding affinity of 627E PB2 to NP was weaker than that of wild type. The binding affinity of 199A+475L PB2 to NP was weaker in the presence of viral-like RNA， but it was stronger in the absence of viral-like RNA, when comparing with wild type. Accordingly, the position 199 and 475 on PB2 could be the crucial determinants to affect vRNP activity through the mechanisms different from the position 627.

目錄
指導教授推薦書
論文口試委員審定書
長庚大學碩博士論文授權書………………………………………………iv
致謝…………………………………………………………………………vi
中文摘要…………………………………………………………………...vii
英文摘要…………………………………………………………………..viii
第一章 簡介……………………………………………………………….1
第二章 研究目的………………………………………………………...10
第三章 實驗材料與方法 ……………………………………………….12
第四章 實驗結果………………………………………………………...20
第五章 討論與未來方向………………………………………………...25
參考文獻 ………………………………………………………………….35
圖表………………………………………………………………………...41
附錄 ……………………………………………………………………….55



圖表目錄
表一 A型流行性感冒病毒蛋白質與其功能……………………………..41
表二 A/chicken/Brescia/1902 (H7N7)、A/Brevig Mission/1/1918 (H1N1) 和A/WSN/33 (H1N1) PB2胺基酸位點之比較...........................................42
表三 A/WSN/33 (H1N1) PB2蛋白質欲突變位置與其胺基酸之對應.....43
表四 進行PB2 avian-like mutations之引子序列………………………...44
表五 H1N1亞型A型流行性感冒病毒PB2之胺基酸序列分析………...45
圖一 以西方墨點法確認PB2蛋白質的表現…………………………….46
圖二 不同avian-like PB2在HEK 293T細胞中降低vRNP的活性……...47
圖三 不同avian-like PB2在DF-1細胞中降低vRNP的活性……………48
圖四 共同表現viral-like RNA的狀況下，觀察在HEK 293T細胞中avian-like PB2和NP的交互作用……………………………………........49
圖五 缺乏viral-like RNA的狀況下，觀察HEK 293T細胞中avian-like PB2和NP的交互作用…………………………………………………….51
附表一 PB2蛋白質中可區分禽流感及人流感病毒的胺基酸位點……..52
附圖一 A型流行性感冒病毒顆粒結構…………………………………..53
附圖二 PB2蛋白質的功能區及和宿主適應有關的胺基酸位點………..54

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Mehle, A., and Doudna, J.A. (2008). An inhibitory activity in human cells restricts the function of an avian-like influenza virus polymerase. Cell host & microbe 4, 111-122.
Subbarao, E.K., London, W., and Murphy, B.R. (1993). A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. Journal of virology 67, 1761-1764.