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研究生:黃博煜
研究生(外文):Huang, Po-Yu
論文名稱:探討A型流感病毒聚合酶第一鹼性蛋白上 216號胺基酸對病毒複製及毒力的影響
論文名稱(外文):Virological Characterization of Residue 216 on PB1 Protein in Replicase of Influenza A Virus
指導教授:廖經倫
指導教授(外文):Liao, Ching-Len
口試委員:林宜玲施信如陳紀如林昌棋
口試委員(外文):Lin, Yi-LingShih, Shin-RuChen, Chi-JuLin, Chang-Chi
口試日期:2012-06-26
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:63
中文關鍵詞:A型流感聚合酶複製毒力
外文關鍵詞:Influenza A virusPolymeraseReplicase
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A型流感病毒屬於正黏液病毒科的病毒,含八段單股負向RNA遺傳體,並帶RNA-dependent RNA polymerase (RdRP)來掌管病毒複製及轉錄。而病毒RdRP 複合體是由PB1、PB2、PA組成,其中PB1具有RdRP用以合成RNA的重要酵素功能。2009年曾有文獻報導PB1第216胺基酸在不同族群的流感病毒中呈現特殊的分佈,如在禽類流感是以Serine為主,豬類流感則Serine及Glycine均分,而人類流感卻以Glycine為主。為進一步探索該位置所扮演的角色,我們採用實驗室級野生型WSN33及PR8和2009年於台灣所分離之豬源流感TW126等病毒株,運用點突變結合反轉遺傳回收病毒的技術,生產各式突變病毒株。我們發現,PB1S216G的胺基酸置換,會造成病毒RdRP在mini-replicon系統中的突變指數 (Accumulative mutation index; AMI)上升;反之,若進行PB1G216S的置換則使AMI降低。而這些PB1的突變病毒株雖然在細胞中的生長狀況類似,但對小鼠的毒力卻有所差異,PB1S216G的置換使毒力下降,反之在豬源流感PB1G216S的置換則導致毒力增強。根據這些結果我們推論,PB1-216位點之胺基酸置換可能影響了病毒RdRP於複製RNA過程中的忠誠度,因此直接或者間接的造成病毒在小鼠體內毒力的差異。
Influenza A virus belongs to Orthomyxoviridae family, which is enveloped, negative-sense RNA viruses with eight segmented genomes. Each RNA genome is packaged into ribonucleoprotein (RNP) complex containing nucleoprotein (NP) and the trimeric RNA-dependent RNA polymerase (RdRP), which is comprises of PA, PB1 and PB2 subunits. Among these, PB1 contains the polymerase catalytic domain which is essential for virus genome replication and mRNA transcription. According to previous epidemiology study, residue 216 on PB1 protein of influenza A viruses isolated from different infected hosts exhibits species-specific signature; that is, serine in avian-isolated influenza virus, either serine or glycine in swine isolates, and predominantly glycine in 2009 human pandemic strains. To further study the potential role of such signatures in virus replication capability, replicase fidelity and virulence in mice, we generated mutant viruses using laboratory influenza A virus strains of WSN33 and PR8, as well as TW126 isolated in Taiwan in 2009. We found that PB1S216G substitution attenuated viral virulence in mice of both WSN33 and PR8, whereas PB1G216S enhanced the virulence of PR8/TW126PB1 reassortment virus. There is no apparent difference in viral growth of these viruses under culture condition. However, we found PB1S216G resulted in higher accumulative mutation index (AMI) of replicase complex assayed by mini-replicon system, and vice versa for lower AMI in PB1G216S mutants. We therefore hypothesized that substitution at this point affects viral virulence in mice through altering influenza RdRP fidelity and revealed a potential role of residue 216 on PB1 for virulence determination.
目 錄
項目 *********************** 頁數
正文目錄............................................................................................................. I
第一章、 導論................................................................................................. 1
第一節、 A型流感病毒............................................................................... 1
第二節、 病毒基因結構及蛋白................................................................... 2
第三節、 病毒生活史及其宿主範圍決定與適應....................................... 3
第四節、 病毒的核糖核酸聚合酶及其特性……………........................... 5
第五節、 簡述流感病毒核糖核酸聚合酶突變頻率檢測平台................... 6
第二章、 研究動機與目的............................................................................. 9
第三章、 材料與方法................................................................................... 10
第一節、 細胞、病毒與實驗動物............................................................. 10
壹、 細胞株............................................................................................... 10
貳、 病毒................................................................................................... 11
叁、 實驗動物........................................................................................... 12
第二節、 實驗方法..................................................................................... 12
壹、 質體DNA的製備............................................................................ 12
貳、 轉染 (Transfection).......................................................................... 13
叁、 雙冷光酶測定 (Dual-Luciferase reporter assay)............................. 13
肆、 反轉遺傳技術 (Reverse-genetics)................................................... 14
伍、 病毒效價測定 (Plaque-forming assay)........................................... 14
陸、 病毒毒力檢測 (Virus virulence test) .............................................. 15
第四章、 結果............................................................................................... 16
第一節、 探討A型流感WSN33病毒株在PB1蛋白216位點上進行胺 基酸置換後所造成的影響......................................................... 16
壹、 於感染細胞中探討此胺基酸置換對WSN33病毒的影響............ 16
貳、 於感染小鼠中探討此胺基酸置換對WSN33病毒毒力的影響.... 16
叁、 於轉染細胞中探討此胺基酸置換對WSN33病毒微複製子的影響……………………………………………………..……..……… 17
第二節、 以TW126病毒株為背景之TW126/PR8HA:NA重組病毒,探討A型流感在PB1蛋白216位點上進行胺基酸置換後所造成的影響................................................................................................. 18
壹、 於感染小鼠中探討此胺基酸置換對TW126/PR8HA:NA重組病毒毒力的影響............................................................................................... 19
貳、 於轉染細胞中探討此胺基酸置換對TW126病毒微複製子的影響…………………………………………………………………... 20
第三節、 探討A型流感PR8病毒株在PB1蛋白216位點上進行胺基酸置換後所造成的影響................................................................. 20
壹、 於感染細胞中探討此胺基酸置換對PR8病毒的影響.................. 21
貳、 於感染小鼠中探討此胺基酸置換對PR8病毒毒力的影響.......... 21
叁、 於轉染細胞中探討此胺基酸置換對PR8病毒微複製子的影響.. 22
第四節、 以PR8病毒株為背景之PR8/TW126PB1重組病毒,探討A型流感在PB1蛋白216位點上進行胺基酸置換後所造成的影響.. 23
壹、 於感染小鼠中探討此胺基酸置換對PR8/TW126PB1重組病毒毒力 的影響.............................................................................................. 24
貳、 於感染細胞中探討此胺基酸置換對PR8/TW126PB1重組病毒的影 響...................................................................................................... 25
叁、 於轉染細胞中探討此胺基酸置換對PR8/TW126PB1重組病毒微複 製子的影響...................................................................................... 25
第五章、 討論.............................................................................................. 28
第六章、 參考文獻...................................................................................... 34
表...................................................................................................................... 37
表 1、 不同流感病毒族群在PB1蛋白216位點上的胺基酸分佈
圖...................................................................................................................... 38
圖 1、 A型流感WSN33和PR8病毒株PB1蛋白胺基酸序列比較
圖 2、 於感染細胞中探討不同PB1蛋白組合的WSN33病毒株之複製能力
圖 3、 A型流感WSN33和TW126病毒株PB1蛋白胺基酸序列比較
圖 4、 於感染細胞中探討不同PB1蛋白組合的PR8病毒株之複製能力
圖 5、 於感染小鼠中探討不同PB1蛋白組合的PR8病毒株之毒力
圖 6、 於轉染細胞中探討不同PB1蛋白組合的PR8病毒微複製子之複製 能力
圖 7、 於轉染細胞中探討不同PB1蛋白組合的PR8病毒微複製子之突變 頻率
圖 8、 於感染小鼠中探討不同PB1蛋白組合的PR8/TW126PB1重組病毒株 之毒力
圖 9、 於感染細胞中探討不同PB1蛋白組合的PR8/TW126PB1病毒株之複 製能力
圖 10、 於轉染細胞中探討不同PB1蛋白組合的PR8/TW126PB1重組病毒微複製子之複製能力
圖 11、 於轉染細胞中探討不同PB1蛋白組合的PR8/TW126PB1重組病毒微複製子之突變頻率
圖 12、 A型流感PR8和TW126病毒株PB1蛋白胺基酸序列比較
附錄................................................................................................................. 50
附錄一、 Dual-luciferase Reporter Gene
附錄二、 於感染小鼠中探討不同PB1蛋白組合的WSN33病毒株之毒力
附錄三、 於轉染細胞中探討不同PB1蛋白組合的WSN33病毒微複製子之 複製能力
附錄四、 於感染小鼠中探討不同PB1蛋白組合的TW126/PR8 HA:NA重組病毒株之毒力
附錄五、 於轉染細胞中探討不同PB1蛋白組合的TW126病毒微複製子之 複製能力

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