魚類神經壞死病毒(Betanodavirus)可以感染多種魚類的仔雉魚且致死率極高，造成養殖業極大的損失。本研究以龍膽石斑神經壞死病毒(DGNNV)的似病毒顆粒(VLPs)做為材料，分析病毒顆粒組裝(assembly)與崩解(dissociation)的特性。以螯合劑、還原劑或是氧化劑進行試驗，結果證實VLPs的組裝只需要鈣離子的參與。利用點突變來分析外殼蛋白序列可能與鈣離子結合的功能域 (motif) DxxDxD，發現其中第133個位置的天門冬氨酸(Aspartic acid, D)對VLPs的影響最大，一旦突變為不帶電的天門冬醯胺(Asparagine, N)，外殼蛋白就無法組裝成VLPs，另外兩個Aspartic acid位置的突變(D130N及D135N)則可以觀察到不規則型態的蛋白質或是似病毒顆粒兩種現象。進一步測試各種VLPs對熱處理的穩定度，結果發現D135N VLPs對溫度的耐受性稍高於野生型VLPs (85℃)，然而D130N VLPs只要超過60℃就會崩解。上述結果推知DxxDxD功能域中三個Aspartic acid對於VLPs的形成與穩定度都十分重要，對鈣離子配位而言，DxxD可能比DxD提供了更穩定的配體(ligand)。

Piscine nodaviruses are members of genus Betanodavirus, which infect more than 30 species of fish and cause massive mortality in larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the dissociation and reassembly of betanodavirus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. The experiments with calcium-chelating or reducing/oxidizing reagents elicited that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. In the mutational analysis of DxxDxD that is putatively involved in the coordination of calcium ions, the results showed that the D133N mutation significantly disrupted the assembly of VLPs while D130N and D135N mutants produced heterogeneous particles with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It is deduced that three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and, among them, the DxxD provides a more stable coordinate of calcium-ligand than DxD.

摘要…………………………………………………………………………..............i
Abstract………………………………………………………………………………ii
目錄…………………………………………………………………………………iii
表目錄……………………………………………………………………………….v
圖目錄……………………………………………………………………….…....vi

第一章、序論………………………………………………………………………..1
第二章、文獻回顧…………………………………………………………………..5
第一節、Betanodavirus宿主、病毒株命名與病徵…………………………..5
第二節、Nodavirus基因序列分析……………………………………………8
第三節、RNA聚合酶研究……………………………………………...……10
第四節、外殼蛋白特性分析……………………………………………........13
第五節、細胞增殖、感染途徑與內化機制…………………………………17
第六節、似病毒顆粒………………………………………………………....20
第三章、病毒顆粒崩解與再組裝的機制………………………………………....27
第一節、前言…………………………………………………………............27
第二節、材料方法………………………………………………………........31
2.1 似病毒顆粒純化及分析…………………………………............31
2.1.1 大腸桿菌表現病毒外殼蛋白………………………...........31
2.1.2 似病毒顆粒純化……………………………………….......32
2.1.3 連續式分光光度計分析…………………………………...32
2.1.4 螯合劑、核醣核酸酵素、氧化劑以及還原劑對似病毒顆粒
影響……………………………………………...................33
2.1.5 蛋白質電泳…………………………………………….......34
2.1.6 蛋白質定量...........................................................................35
2.1.7 Anti-VLP 抗血清的製備與純化……………………….....35
2.1.8 西方墨點法…………………………………………….......36
2.1.9 圓雙色光譜測定………………………...............................37
2.2 細胞培養與病毒增殖……………………………………………37
2.2.1 無菌操作基本技術………………………………………...37
2.2.2 細胞繼代……………………………………………...........38
2.2.3 細胞保存……………………………………………...........38
2.2.4 冷凍細胞活化……………………………………………...39
2.2.5 病毒增殖……………………………………………...........40
2.2.6 病毒純化……………………………………………...........40

2.3 電子顯微鏡樣品觀察…………………………………………....41
2.3.2 樣品負染色…………………………………………….......41
2.3.3 蛋白質樣品觀察……………………………………….......41
第三節、結果………………………………………………………...............42
3.1 DGNNV VLPs純化…………………………………..................42
3.2 螯合劑對似病毒顆粒的影響………………………....................43
3.3 核醣核酸酶對似病毒顆粒的影響………………………............45
3.4 雙硫鍵對似病毒顆粒的影響………………………....................45

3.5 鈣離子參與似病毒顆粒的重組裝………………………............47
3.6 二價離子對VLP重組裝的影響………………………...............48

第四節、討論………………………………………………………...............51
第四章、神經壞死病毒顆粒鈣離子模體的分析……………….......................56
第一節、前言...................................................................................................56
第二節、材料方法...........................................................................................59

2.1 質體抽取…………………………………....................................59
2.2 氨基酸定點突變............................................................................59
2.3 勝任細胞製作................................................................................60
2.4 細胞轉型........................................................................................61

2.5 似病毒顆粒 RNA 抽取................................................................61
2.6 RNA 電泳......................................................................................61

2.7 突變株似病毒顆粒純化及分析....................................................62
第三節、結果...................................................................................................63

3.1 病毒外殼蛋白鈣離子模體序列的分析....................................63

3.2 DxxDxD模體點突變分析.............................................................64

3.3 突變株的似病毒顆粒對溫度穩定度............................................68
3.4 螯合劑對突變株似病毒顆粒的影響............................................69
第四節、討論………………………………………………………...............71

第五章、結論...........................................................................................................79
參考文獻...................................................................................................................81

圖表.........................................................................................................................101
附錄.........................................................................................................................152

附錄A. Nodavirus序列整理與比對.............................................................153
附錄B. 突變株序列.......................................................................................158

附錄D. 實驗步驟...........................................................................................170
附錄D. 參考文獻圖表...................................................................................178
附錄D. 個人著作...........................................................................................182

表目錄
Table 1. Alignment of calcium-binding motifs in viral capsid proteins.................102
Table 2. The list of primers.....................................................................................105


圖目錄
Fig. 1. Flowchart of sediment analysis of VLP.......………………………….......106
Fig. 2. Purification and anylisis of DGNNV VLPs...…………………………….107
Fig. 3. Electron micrograph of VLPs that assembled in E. coli expressing the
wild-type capsid protein of DGNNV……………………………………...108
Fig. 4. Sedimentation profiles of DGNNV and VLPs treated with chelating agents
..............................................................................................109
Fig. 5. The SDS-PAGE analysis of untreated and EGTA-dissociated VLPs..........111
Fig. 6. Electron micrographs of wild-type and EGTA-dissociated VLPs................112
Fig. 7. Sedimentation profile of DGNNV VLPs treated with RNase......................113
Fig. 8. Sedimentation profiles of DGNNV VLPs treated with reducing agents......114
Fig. 9. Sedimentation profile of DGNNV VLPs treated with diamide..................115
Fig. 10. Sedimentation profile and SDS-PAGE analysis of reassembled VLPs......116
Fig. 11. Electron micrographs of calcium-reassembled VLPs................................117
Fig. 12. Dissociation and reassembly of DGNNV VLPs........................................118
Fig. 13. Immunoblotting analysis of EGTA-dissociated VLPs...............................119
Fig. 14. Circular dichroism spectropolarimeter analyses for DGNNV VLPs.........120
Fig. 15. VLPs reassembled by alkaline metal ions..................................................121
Fig. 16. Electron micrographs of VLPs reassembled by alkaline metal ions..........122
Fig. 17. VLPs reassembled by transition metal ions...............................................123
Fig. 18. Electron micrographs of VLPs reassembled by transition metal ions.......124
Fig. 19. The calcium-binding motif of TBSV.........................................................125
Fig. 20. Alignment of DGNNV and TBSV coat protein sequences.......................126
Fig. 21. The calcium-binding motif of BBV...........................................................127
Fig. 22. Multiple alignment of amino acid sequences of the betanodavirus coat
proteins.......................................................................................................128
Fig. 23. Sedimentation profile and SDS-PAGE analysis of D130N VLPs.............131
Fig. 24. Electron micrographs of D130N VLPs......................................................132
Fig. 25. Sedimentation profile and SDS-PAGE analysis of D133N VLPs.............133
Fig. 26. Electron micrographs of D133N VLPs......................................................134
Fig. 27. Sedimentation profile and SDS-PAGE analysis of D135N VLPs.............135
Fig. 28. Electron micrographs of D135N VLPs......................................................136
Fig. 29. Sedimentation profiles and SDS-PAGE analysis of mutant VLPs............137
Fig. 30. RNA analysis of wild-type and mutant VLPs............................................138
Fig. 31. Sedimentation profiles of thermal stability of wild-type VLPs.................139
Fig. 32. Electron micrographs of thermal stability of wild-type VLPs...................140
Fig. 33. Sedimentation profiles of thermal stability of D130N VLPs.....................143
Fig. 34. Electron micrographs of thermal stability of D130N mutant.....................144
Fig. 35. Sedimentation profiles of thermal stability of D135N VLPs.....................145
Fig. 36. Electron micrographs of thermal stability of D135N mutant.....................146
Fig. 37. Disassembly of D130N and D135N VLPs.................................................149
Fig. 38. Hypothetical scheme: dual mode of calcium-ligand interactions for virion
formation...................................................................................................150

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