在全世界各地有許多養殖魚類遭受到神經壞死病毒 (dragon grouper nervous necrosis virus, DGNNV; Betanodavirus) 的感染而造成經濟上的嚴重損失。魚類神經壞死病毒會引發 40 種以上魚類的神經壞死症，其常見的病徵為不正常游動和體色變黑，死亡率最高可達 100%，因此，預防病毒與控制疫情的發展是很重要。
Betanodavirus 含有兩條 RNA，稱為 RNA1 及 RNA2，所轉譯的蛋白質分別為 RNA 聚合酵酶及外殼蛋白。本研究以龍膽石斑神經壞死病毒似病毒顆粒 (virus-like particles, VLPs) 為材料，分析外殼蛋白形成結晶的特性。利用大腸桿菌 (E. coli) 表現系統中大量表現生產外殼蛋白，經由純化過程所得到的高純度 DGNNV VLPs 蛋白質，並以蒸氣擴散法來篩選適合的結晶條件，可以成功得到直徑約 0.22-0.27 mm 的菱形六面體的 DGNNV VLPs 晶體，此晶體在低溫下進行 X-ray 繞射，可以得到解析度約 7.5 Å 的完整晶體繞射圖，經由計算的結果，其晶體屬於 R32 group，unit-cell parameters 為 a = b = 353.00 Å, c = 800.40 Å, α = β = 90°, γ = 120°。
單一的 DGNNV VLPs 晶體 X-ray 繞射點以每 60° 衍射範圍，0.2° 擺動角度及曝光 30 sec 的方式收集，總計有 299 張 X-ray 繞射點圖，完整度達到 93.2%。利用 CCP4 的 POLARRFN 程式分析 DGNNV VLPs 晶體的 self-rotation function 圖，證實可以得到 DGNNV VLPs 晶體的二十面體之五重對稱 (five-fold)、三元對稱與二元對稱，其 κ 值分別為 72°，120° 與 180°。進一步分析 DGNNV VLPs 晶體的 X-ray 繞射數據與已解出的 3D 圖像重建龍膽石斑魚神經壞死病毒的結構。本研究 DGNNV VLPs 晶體結構為目前首見的成就對於其它與此相近似的魚類神經壞死病毒結構，將有更深入了解，並擴大相關研究的視野。

The Betanodaviruses caused nerve necrosis in more than 40 species. The clinical symptoms of virus-infected fish include abnormal movement and darkness in body color, with mortality as high as 100%. To prevent and control the outbreak of NNV is a very important issue.
There are two RNAs in the Betanodaviruses. RNA1 encodes RNA dependent RNA polymerase and RNA2 encodes capsid protein. To study the infection process of dragon grouper nervous necrosis virus (DGNNV), native virus and E. coli-produced virus-like particles (VLPs), and analysis revealed different impacts on VLP formation crystallization. Virus-like particles are formed in E. coli expressing the full-length ORF encoding the DGNNV capsid protein. The native protein was purified and crystallized by vapor diffusion method against mother liquor. The crystals grew to the size of 0.22-0.27 mm in one week and diffracted by X-rays to 7.5 Å resolution. The data were indexed in a primitive rhombohedral crystal system. Preliminary processing of the DGNNV VLPs diffracting data suggests that the crystal belong to space group R32; a = b = 353.00 Å, c = 800.40 Å, α = β = 90°, γ = 120°.
A total of 299 images were collected from a single VLP crystal by 60° diffracting range, 0.2° oscillation angle, and 30s exposure. 60,035 observed reflections were reduced to 23,268 unique reflections with an overall Rmerge of 18.2% and a completeness of 93.2%. Self-rotation function maps were calculated using the program POLARRFN from the CCP4 suite; spherical angles of κ = 72°, 120°, and 180° were for five-fold, three-fold and two-fold axes of icosahedral symmetry. This study is the first report about X-ray diffractometer the crystal of DGNNV capsid protein, which provides an example of DGNNV threedimensiond structure for other related Betanodaviruses.

目錄
頁次
論文審定書…………………………………………………………........ i
誌謝…………………………………………………………………........ ii
摘要…………………………………………………………………........ iii
Abstract………………………………………………………………..... v
目錄…………………………………………………………….............. vii
表目錄……………………………………………………………........... xiii
圖目錄………………………………………….................................. xiv
第一章、緒論………………………………………………………….... 1
第二章、文獻回顧…………………………………........................... 5
一、Betanodavirus 的發現、病毒株的命名與病徵………………...... 5
二、Betanodavirus 所感染的魚種與地理分佈…………………......... 7
三、Betanodavirus 的分子生物學特性…………………………......... 10
四、Betanodavirus 感染的症狀…………………………………......... 11
五、Betanodavirus 的偵測方法…………………………………......... 12
六、細胞培養增殖 Betanodavirus 的細胞株……………………........ 17
七、Betanodavirus 的致病力……………………………………......... 22
八、疫苗的發展…………………………………………………… ........27
第三章、龍膽石斑神經壞死病毒似病毒顆粒的結晶與X-ray繞射....... 31
壹、前言……………………………………………………………….... 31
一、Betanodavirus 的基因序列與分類…………………………......... 31
二、Betanodavirus 的結構與穩定性……………………………......... 33
三、似病毒顆粒…………………………………………………… ........35
四、研究目的………………………………………………………...... 41
貳、材料與方法……………………………………………………….... 42
一、SSN-1 細胞培養與病毒增殖……………………………….......... 42
1. 無菌操作基本技術………………………………………….............. 42
2. SSN-1 細胞的繼代…………………………………………............. 42
3. SSN-1 細胞的保存與冷凍的細胞活化……………………............. 44
4. 利用 SSN-1 細胞增殖 DGNNV…………………………................ 45
5. 純化 DGNNV……………………………………………….............. 46
二、DGNNV VLPs 樣品的純化與分析…………………………......... 47
1. 大腸桿菌表現 DGNNV VLPs 的外殼蛋白……………….............. 47
2. DGNNV VLPs 的純化-蔗糖梯度離心法………………….............. 49
3. DGNNV VLPs 的純化-氯化銫梯度離心法……………….............. 51
4. 連續式分光光度計分析…………………………………….............. 53
5. 蛋白質定量-Lowry 法……………………………………................ 55
6. 蛋白質的電泳分析………………………………………….............. 55
7. 抗 VLPs 的小鼠血清製備與純化…………………………............. 57
8. 西方墨點法 (Western blotting) ……………………………............ 58
9. 抽取質體 DNA……………………………………………................ 60
10. DNA 的電泳分析………………………………………….............. 61
11. 抽取膠體 DNA 與純化……………………………………............. 62
12. 勝任細胞 (Competent cell) 的製備………………………............ 63
13. 細胞轉型…………………………………………………............... 63
14. 抽取 VLPs 的 RNA……………………………………................. 64
15. RNA 的電泳分析………………………………………….............. 64
三、穿透式電子顯微鏡的觀察 ………………………………….......... 65
1. 蛋白質樣品的負染色 (Negative stain) ……………………............ 65
2. 蛋白質樣品的觀察………………………………………….............. 66
四、蛋白質的結晶實驗與過程…………………………………… ........66
1. 結晶的方法 (Crystallization) ………………………………............ 66
2. 結晶條件的篩選…………………………………………….............. 69
3. 結晶條件的最佳化 (Optimization) ……………………….............. 70
4. 篩選適合的抗凍劑 (Cryo-protectant) ……………………............. 71
5. 添加物 (Additive) 的試驗…………………………………............... 71
6. 晶種誘導晶體生長的試驗………………………………….............. 72
7. 晶體的判斷與挑晶體的方法……………………………….............. 73
五、利用 X-ray 晶體的繞射技術來解析蛋白質的結構………........... 74
1. 晶體的繞射前的準備……………………………………….............. 74
2. 單晶的繞射實驗…………………………………………….............. 76
3. 相位 (Phase) 的判定………………………………………............. 77
4. 以 X-ray 來決定蛋白質的結構…………………………….............. 79
參、結果……………………………………………………………….... 81
1. DGNNV 的增殖與純化…………………………………….............. 81
2. DGNNV 似病毒顆粒的純化……………………………….............. 83
3. 野生型似病毒顆粒的熱穩定性影響……………………….............. 87
4. DGNNV VLPs 結晶條件的初步篩選………………………............ 88
5. DGNNV VLPs 結晶條件的進一步篩選……………………............ 90
(1) 添加不同濃度的 NaCl…………………………………….............. 90
(2) 添加不同百分比濃度的Glycerol作為一抗凍劑…….................... 91
(3) 提高Glycerol百分比濃度作為一抗凍劑與結晶培養溫度.............. 91
6. 以染劑染色蛋白質晶體…………………………………….............. 93
7. 添加二價金屬離子對蛋白晶體形成的影響………………..............93
(1) 添加鹼土金屬……………………………………………................ 93
(2) 添加過渡元素……………………………………………................ 95
8. X-ray 晶體繞射初步數據…………………………………............... 96
肆、討論……………………………………………………………….... 98
第四章、DGNNV 外殼蛋白 N 端與 C 端結晶特性的探討……......... 104
壹、前言……………………………………………………………….... 104
貳、材料與方法……………………………………………………….... 105
1. DGNNV VLPs 純化步驟的修正……………………………............ 105
2. DGNNV VLPs 純化過程中改變緩衝液與 pH 值………............... 108
3. poly-His tagged 選植株外殼蛋白純化……………………............. 109
4. 負染 (Negative stain) 及電子顯微鏡……………………............... 110
參、結果……………………………………………………………….... 111
1. DGNNV VLPs 純化過程分別以Detergent、Enzyme 以及
Chloroform 處理的影響………………………..............................111
2. poly-His tagged 選植株外殼蛋白純化與結晶……………............. 115
肆、討論……………………………………………………………….... 117
第五章、結論………………………………………………………….... 120
第六章、參考文獻…………………………………………………........ 121
第七章、圖表………………………………………………………........ 149
第八章、附錄………………………………………………………........ 199
Appendix A. Crystallization Conditions for Intact Viruses,
Subassemblies, Structural Proteins, and Their Complexes…….........................................................................199
Appendix B. The crystal screen from Academia Sinica………....... 205
Appendix C. Components of Kits and screening conditions for
DGNNV VLP…………………………………………………................213
Appendix D. Data-processing statistics for Alphanodavirus and
Betanodavirus from RCSB Protein Data Bank, Academia Sinica,
and NSRRC……………………………………………....................... 217
Appendix E. 選殖質體圖與其序列……………………………............ 218
附錄 F、個人著作……………………………………………………..... 232

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