魚類神經壞死病毒(Betanodavirus)會引發多種魚類的神經壞死，Betanodavirus含有兩條RNA，稱為RNA1及RNA2，所轉譯的蛋白質分別為RNA聚合酵素(RdRp; RNA dependent RNA polymerase)及外殼蛋白。本研究分析了兩種石斑魚類神經壞死病毒外殼蛋白基因，分別為來自瑪拉巴石斑的MGNNV及龍膽石斑的DGNNV，以序列相似度來看，兩種石斑病毒相似程度高達99%，而與SJNNV及DlEV則分別為87.8%和78.6%。所選殖的外殼蛋白基因經由表現性的質體上T5啟動子表現後，可以組裝成似病毒顆粒。
截短外殼蛋白N端及C端氨基酸有助於了解其所代表的功能，截短35及52個氨基酸將完全造成似病毒顆粒組裝能力的喪失，可能為RNA結合能力所造成的影響，而將外殼蛋白的N端分別作4, 16, 25,個氨基酸截短，似病毒顆粒組裝能力依然存在，而C端則僅僅只能截短3個氨基酸不會影響似病毒顆粒的組裝，顯示C端氨基酸敏感性比N端氨基酸高。
利用點突變來探討Aspartic acid對似病毒顆粒結構的影響，其中D75突變成N75後，形成似病毒顆粒的能力消失，但三倍體依然存在，可能是三倍體之間的鍵結遭受到破壞而導致此一現象。D54突變成N54後，所形成的似病毒顆粒對於RNA結合能力降低。D335突變之後，會大量減低似病毒顆粒形成的能力，這樣的結果可能與突變的似病毒顆粒組成穩定性有關。

The Betanodaviruses caused nerve necrosis in several fishes. There are two RNAs in the Betanodaviruses. RNA1 encodes RNA dependent RNA polymerase and RNA2 encodes capsid protein. I analyzed the RNA2 sequences of the viruses isolated from two species of grouper, Epinephelus malabaricus (MGNNV) and E. lanceolatus (DGNNV). The similarity of two grouper viruses (GNNV) was 99%. Their similarities to DlEV and SJNNV were 87.8% and 78.6%, respectively. The capsid protein was successful expressed and assembled to virus-like particles.
Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively.
Expression the ORF of RNA2 in E. coli formed virus-like particles, indistinguishable from native virus particles in appearance, whereas a mutant of Asp-75 expressed no VLPs. The emergence of a trimer band in mutant D75N as wild type suggested that the Asp-75 mutation could halt the packaging process in the trimer stage, not proceeding to assemble intact VLPs. The D54N mutant remained the ability of VLP formation but lost packaging high molecular weight of RNAs. Another Asp mutant at C-terminus, D335A, lost the ability of VLP assembly. The D335 may play an important role on the instability of VLP structure.

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
第一章、緒論…………………………………………………………….5
第二章、文獻回顧………………………………………………………..7
第一節、 魚類病毒…………………………….……………….…7
第二節、 Betanodavirus……………………….…………………..8
第三節、 增殖Betanodavirus…………………....………………18
第四節、 魚類病毒接受子…………………….………...……….21
第五節、 似病毒顆粒…………………………...………………..24
第三章、材料與方法…………………………………………….……..44
第一節、 石斑神經壞死病毒篩選……………………..…….…44
第二節、 DNA操作技術………………………………..……….46
第三節、 似病毒顆粒表現及分析……………………………….51
第四節、 選殖株………………………………………………….58
第四章、石斑病毒RNA2基因體分析………………………….…….101
第一節、 前言…………………………………………………...101
第二節、 材料與方法…………………………………………...105
第三節、 結果與討論………………………………………..….106
4.1 RT-PCR偵測病毒檢體…………………………….…106
4.2分析病毒RNA2…………………………………..……107
第五章、以大腸桿菌表現DGNNV似病毒顆粒………………….…123
第一節、前言………………………………………………….123
第二節、材料與方法………………………………………….127
第三節、結果與討論………………………………………….128
5.1完整外殼蛋白基因之表現……………………….……128
5.2 C 端蛋白質片段的表現……………………...……….129
5.3去除前52個氨基酸的蛋白質表現………………..….130
5.4構築大量表現的病毒外殼蛋白質體……………….…131
5.5溶氧對蛋白質表現的影響…………………………….132
5.6培養溫度對似病毒顆粒產生的影響……………….…133
5.7不同Sucrose 濃度對似病毒顆粒純化的影響………...134
5.8不同緩衝溶液對VLP的影響……………………….….135
5.9利用CsCl 做進一步的純化……………………………136
5.10似病毒顆粒的表現條件的最佳化……………………137
5.11不同IPTG濃度對外殼蛋白表現的影響…………...…139
第六章、DGNNV外殼蛋白N端及C端特性之探討………..………...169
第一節、 前言……………………………………………………169
第二節、 材料與方法……………………………………..……172
第三節、 結果與討論…………………………………………..173
6.1 N端外殼蛋白的截短……………………………..….173
6.2 C端外殼蛋白的截短………………………………..175
6.3 RNA含量……………………………………………176
第七章、Aspartic acid點突變對似病毒顆粒的影響………………...186
第一節、 前言……………………………………………….…186
第二節、 材料與方法………………………………………….188
第三節、 結果與討論……………………………………….…189
7.1 Asp 54及Asp 75點突變對病毒顆粒形成的影響…189
7.2 D335的點突變……………..…………………..……191
第八章、結論………………………………………………………..…205
參考文獻………………………………………………………………207
附錄、A選殖株序列圖……………………………………………….230
附錄、B 著作…………………………………………………...…….317

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