Nodaviridae科分為alphanodaviruses和betanodaviruses兩種病毒。他們分別感染昆蟲及魚類。在我們的研究中，以大腸桿菌表現DGNNV的病毒外殼蛋。我們突變外殼蛋白N端的精氨酸，以研究29-31號位置的精氨酸所扮演的角色。當截去25個在N端的外殼蛋白氨基酸時，只要將任兩個在29-31號位置的精氨酸突變為丙氨酸，會使得似病毒顆粒無法形成。另一方面，在23-25號位置延長3個精氨酸並不會增強RNA包裹進入似病毒顆粒中的量。此外，如同野生型似病毒顆粒，N端截短突變的似病毒顆粒都具RNase A抵抗性，但其產量就明顯少於野生型似病毒顆粒。在單點丙氨酸突變株中，R29A的似病毒顆粒明顯的高於其它兩個突變株 (R30A和R31A)。使用圓二色光譜觀察DGNNV似病毒顆粒的熱變性過程及熱穩定性，我們發現突變23-31號位置的精氨酸並不會影響似病毒顆粒的Tm值 (約60℃)。這些發現建議突變似病毒顆粒23-31號位置的精氨酸，不會影響其對RNase A的耐受性和熱穩定性，但會使產量降低。另外，第30和31號位置的精氨酸比第29號位置的精氨酸在似病毒顆粒形成方面來的重要。

Dragon grouper nervous necrosis virus (DGNNV), a betanodavirus, is the causative agent of viral nervous necrosis (VNN) in dragon grouper (Epinephelus lanceolatus). In our study, capsid protein of DGNNV was expressed in Escherichia coli. We mutated arginines at N-termini capsid protein to investigate the role of arginines at 29-31th position. When capsid protein lost 25 amino acids at N-termini VLPs form, mutation in any two arginines at 29-31 position to alanine could the prohibit VLPs formation. Another extending three arginines at 23-25 position wouldn’t increase the RNA encapsulation into VLPs. Furthermore, N-termini mutated VLPs were all RNase resistance like wt-VLPs, but the yield was distinctly less than wt-VLPs. In the single point alanine mutations, the VLPs yield of R29A was apparently higher than others (R30A and R31A). Using circular dichroism to observe the thermal denature process and thermal stability of DGNNV VLPs, we found the Tm about 60℃ of VLPs wouldn’t alter even if arginines at 23-31 position were mutated. The findings suggested the VLPs of mutated arginines at 23-31 position wouldn’t affect RNase resistance and thermal stability, but the yield were lower. Another, the arginines at the 30 and 31 position is more important than at 29 position for formation of VLPs.

中文摘要............................................................................................ I
Abstract…………………………………………………………....... II
目錄.................................................................................................... III
表目錄................................................................................................ VI
圖目錄................................................................................................ VII
壹、前言............................................................................................ 1
一、Nodavirus............................................................................................... 1
二、Alphanodaviruses life cycle................................................................... 3
三、Alphanodaviruses基因體..................................................................... 6
四、Betanodavirus的分類及感染的宿主................................................... 9
五、Betanodavirus的外觀........................................................................... 11
六、病毒性神經壞死症 (viral nervous necrosis, VNN) 的臨床症狀....... 11
七、RNA在病毒顆粒中的功能.................................................................. 12
八、病毒包?傕NA的選擇性..................................................................... 14
九、病毒組裝的模型.................................................................................. 16
十、不同物理、化學條件下對betanodavirus感染力的影響.................. 18
十一、圓二色性 (circular dichroism) 的原理和應用.............................. 20
十二、似病毒顆粒 (virus-like particles) 的應用...................................... 22
貮、材料與方法................................................................................ 24
一、質體的萃取........................................................................................... 24
二、DNA電泳............................................................................................. 24
三、DNA膠體純化..................................................................................... 25
四、勝任細胞 (competent cell) 的製作..................................................... 26
五、細胞轉型............................................................................................... 26
六、大腸桿菌表現病毒外殼蛋白............................................................... 27
七、病毒外殼蛋白純化............................................................................... 27
八、連續式分分層儀................................................................................... 28
九、蛋白質濃度定量................................................................................... 28
十、蛋白質電泳........................................................................................... 29
十一、圓二色光譜....................................................................................... 30
十二、選殖株設計....................................................................................... 31
參、結果............................................................................................ 37
一、Nodaviruses病毒外殼蛋白N端序列的比對..................................... 37
二、N端正電荷氨基酸的點突變選殖株................................................... 37
三、選殖株生產VLPs之能力分析............................................................. 40
四、選殖株VLPs組成分析........................................................................ 41
五、RNase A處理的選殖株VLPs組成分析............................................. 44
六、似病毒顆粒的相對產量....................................................................... 45
七、似病毒顆粒在長時間加熱下的熱穩定性和二級結構的變化............ 45
八、連續加熱速率的設定........................................................................... 48
九、似病毒顆粒在連續瞬時增溫下的熱穩定性....................................... 48
十、似病毒顆粒在不同波長下的Tm值.................................................... 54
肆、討論............................................................................................ 55
一、外殼蛋白N端影響似病毒顆粒形成的部位..................................... 55
二、比較全長型和N端截短的似病毒顆粒在長時間加熱下的熱穩定性和二級結構變化上的差異...................................................................61
三、突變的似病毒顆粒之熱穩定性.....................................................63
伍、參考文獻.................................................................................... 66
陸、圖表........................................................................................... 76
附錄A................................................................................................ 126
附錄B................................................................................................ 156
附錄C................................................................................................ 157
附錄D................................................................................................ 158
附錄E................................................................................................. 160
附錄F................................................................................................. 161
附錄G................................................................................................. 162
附錄H................................................................................................. 166
附錄I.................................................................................................. 180
附錄J.................................................................................................. 187
附錄K................................................................................................. 188

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