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系統識別號 U0026-2307201613072700
論文名稱(中文) 流感病毒核蛋白K31R及S450G突變對病毒複製的影響
論文名稱(英文) Effect of K31R and S450G Substitutions of Influenza A Nucleoprotein on Viral Replication
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 104
學期 2
出版年 105
研究生(中文) 徐尹美
研究生(英文) Yin-Mei Hsu
學號 T36034084
學位類別 碩士
語文別 英文
論文頁數 66頁
口試委員 指導教授-王貞仁
口試委員-張堯
口試委員-凌斌
口試委員-蕭璦莉
中文關鍵字 A型流感  核蛋白  演化  K31R  S450G  生長特性 
英文關鍵字 Influenza A virus  NP  K31R  S450G  growth property  evolution 
學科別分類
中文摘要 A型流感病毒會造成嚴重的大流行和每年的季節性流行,對全球人類的健康是一項巨大威脅。在流感病毒的八段負股的基因片段中,核蛋白 (NP)具有多種功能,可以形成同源寡聚體(homo-oligomers)、包裹住病毒RNA片段來維持構造穩定、並參與在病毒的複製和轉錄。我們從成大醫院取得A型流感主要流行的亞型H3N2的臨床病毒株,並藉由定序從中找出1999至2015年核蛋白基因的突變點。為了瞭解這些突變點所帶來的影響,我們利用微基因體實驗(mini-genome assay)來檢測病毒的聚合酶活性並找到重要位點。在展現最高聚合酶活性的病毒株Taiwan/N1215/07中,我們發現了在核蛋白上S450G的取代會增加聚合酶的活性,然而K31R會調降它的活性。我們進一步利用逆向遺傳學系統 (reverse genetics system)建構出病毒來確認生長特性。生長曲線、RNA定量和轉譯蛋白量的結果都和聚合酶活性的結果相似,這說明了這兩個位點會去影響病毒複製、轉錄、轉譯。進一步我們發現K31R會造成NP蛋白降解並影響到其他聚合酶蛋白的平衡。綜合以上結果,NP在演化中會透過平衡蛋白的穩定與不穩定來影響病毒特性,我們證明了在核蛋白上的K31R和S450G會改變流感的功能性特性,研究結果還說明這兩個位點的取代可能在病毒演化中的上位關係(epistasis)扮演角色。
英文摘要 Influenza A viruses cause severe pandemics and epidemics annually and pose a great global health threat. Among 8 RNA segments of viral genome, the multiple functions of NP are to form homo-oligomers, encapsidate the virus genomes to maintain the structure and play roles in viral replication and transcription. Previously, we have sequenced clinical isolates of H3N2 from National Cheng Kung Hospital, and identified substitutions in NP from 1999 to 2015. To understand the effects of genetic variations on NP, we used the mini-genome assay to examine the polymerase activity and identified mutations. Among substitutions of Taiwan/N1215/07, which displayed highest polymerase activity, we found that S450G of NP gene increased the polymerase activity but K31R decreased it. In addition, we generated infectious viruses by reverse genetics system and examined their growth properties. Results of growth curve of viral titer, quantitative detection of RNA level and translational protein amount displayed the similar findings as polymerase activity assay. Thus, we demonstrated two substitutions affected the viral replication, transcription, translation. Furthermore, results revealed that K31R may regulate protein degradation and impairing the balance of polymerase proteins. Taken together, NP protein alterations may due to counterbalancing stabilizing and destabilizing which may have the great impact on viral properties. Substitutions K31R and S450G of NP contribute to functional change of influenza virus and may play the roles in epistasis in the viral evolution.
論文目次 Chinese Abstract I
Abstract II
Acknowledgments III
Table of contents IV
List of tables VI
List of figures VII
List of appendix figures VIII
Abbreviations IX
Chapter 1 Introduction 1
1.1 Introduction of Influenza viruses 1
1.1.1 Classification and clinical manifestations of influenza viruses .. 1
1.1.2 Structure of influenza A virus 1
1.1.3 Epidemiology of influenza A virus 1
1.1.4 Evolution of influenza A viruses 2
1.2 Role of gene segments and translated proteins in influenza virus life cycle 3
1.2.1 Virus entry 3
1.2.2 Transcription, Replication and Translation of virus 4
1.2.3 Assembly and viral particle budding 4
1.3 Significance of genetic changes in influenza A viruses 5
1.3.1 Envelope proteins alteration 5
1.3.2 Internal gene evolution 6
1.4 Character of NP in influenza A viruses 7
1.4.1 Structure, Sequence and functional domains of NP 7
1.4.2 Multi-functional roles of NP 7
1.5 Specific aims 8
Chapter 2 Materials and Methods 10
2.1. Materials 10
2.1.1 Cell lines 10
2.1.2 Competent cell 10
2.1.3 Primers for NP expression plasmids construction 10
2.1.4 Primers and Probes for quantitative RT-PCR 10
2.1.5 Plasmid 11
2.1.6 Enzymes 11
2.1.7 Chemical and reagents 12
2.1.8 Buffers and solutions 14
2.1.9 Antibody 15
2.1.10 Kits 15
2.1.11 Instruments 16
2.2. Methods 17
2.2.1 Cell lines and virus isolates 17
2.2.2 Sequencing of NP gene 17
2.2.3 Phylogenetic and amino acid substitution analysis 17
2.2.4 Mini-genome assay 18
2.2.5 Construction of NP expression plasmids 18
2.2.6 Production of viruses by reverse genetics 18
2.2.7 Immunofluorescence stain (IF stain) 19
2.2.8 Virus growth kinetics 19
2.2.9 Plaque assay 19
2.2.10 Quantitative RT-PCR assays for vRNA and mRNA 20
2.2.11 Plasmid transfection and immunoblot analysis 20
2.2.12 Virus infection and immunoblot analysis 21
2.2.13 Virus purification and immunoblot analysis 21
2.2.14 Measuring of protein degradation 21
Chapter 3 Results 23
3.1. Identification of important substitutions from the evolution of NP gene of influenza H3N2 viruses at NCKUH from 1999 to 2015 23
3.1.1 Sequence and phylogenetic analysis of NP gene evolution 23
3.1.2 Substitutions of K31R and S450G of NP affected the polymerase activity. 23
3.2. Determination of the effects of two K31R and S450G on viral replication by recombinant viruses 25
3.2.1 Generation of viruses by reverse genetics system 25
3.2.2 K31R and S450G substitutions affected viral growth properties 26
3.2.3 Two substitutions affected the viral replication, transcription and translation 26
3.2.4 K31R may regulate protein degradation and impairing the balance of polymerase proteins. 27
Chapter 4 Discussion and Conclusions 30
References 37

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