臺灣博碩士論文加值系統

傳染性華氏囊病毒（Infectious bursal diseasse virus, IBDV）主要感染幼雞的未成熟B 淋巴細胞，導致雞隻免疫力下降以及死亡，此疾病對全世界畜牧業造成巨大的經濟損失。於先前研究發現，由傳染性華氏囊病毒殼蛋白所組成的VP2 次病毒顆粒(subviralparticles, SVPs)，其暴露於顆粒外側第249 與253 之兩個histidine 胺基酸殘基（side chain）會與Ni-NTA 吸附，故可藉此固定化金屬親和性管柱進行VP2 次病毒顆粒純化。由於IBDV表面全由VP2 所組成的T=13 正二十面體，故推測固定化金屬親和性管柱可應用於傳染性華氏囊病毒的純化上。本研究利用DF-1 細胞進行病毒之增殖研究，發現以MOI=0.001 感染DF-1 細胞48 小時後，其增殖之病毒力價≧108 pfu/ml。將病毒液經超高速離心沉降後，再佐以固定化金屬親和性
管柱純化，病毒可於30 mM imidazole 緩衝液被充提出，純度約95%，回收率則為9%。經穿透式電子顯微鏡觀察，於純化的病毒顆粒中，除含有粒徑約60 nm 的病毒顆粒外，另具有粒徑約45 nm 的病毒顆粒存在其中。總結本篇研究，我們發表了一種新的傳染性華氏囊病毒純化方法，可應用於後續病毒感染細胞的機制研究上。

Infectious bursal disease virus (IBDV) can infect two weeks old chicken.It causes the necrosis of bursa of Fabricius in infected chicken and results in a severe immunosuppression. IBDV causes great econimical losses to the poultry industry worldwide. In the previous study, we found that IBDV VP2 subviral particles (SVPs) can interact with Ni-NTA due to two surface histidines at amino acids 249 and 253. Thus, the VP2 SVP could be purified with immobilized metal ion affinity chromatography. Interestingly, the IBDV virion was composed of VP2 capsid protein on the surface in a T=13 mannerstructure. Therefore, the IMAC method could be applied to purify IBDV virions. In this study, we amplified IBDV in DF-1 cell, one kind of chicken embryo fibroblast cell line. Virus can amplify over 108 pfu/ml with MOI=0.001 after 48 hours post-infection. Here we report one novel method that involves the use of concentration of virus by ultracentrifugation followed by IMAC. After eluted with 30 mM imidizale, the recovery of IBDV was 7% constrast to crude lysate, and the purity reached 95 %. With TEM analysis, the typical virion particles in 60 nm diameter could be revealed and accompanied with some other particles in 45 nm diameter. In this study, we developed a new viral purification method. Futhermore, the purified virus can be applied for further investigation on viral penetration and replication.

中文摘要 ..................................................................................................... i
Abstract ...................................................................................................... ii
目錄 ........................................................................................................... iii
第一章 文獻回顧 ...................................................................................... 1
ㄧ、傳染性華氏囊病毒 (Infectioous Bursal Disease Virus) ........... 1
（一）甘保羅病（Gumboro disease） .................................... 1
（二）IBDV 的構造與分子生物學上的組成 ......................... 1
（三）免疫原性及致病性 ........................................................ 2
（四）病毒增殖 ........................................................................ 3
（五）病毒的純化方法 ............................................................ 3
（六）VP2 次病毒顆粒與Ni-NTA 的吸附特性 ..................... 4
（七）近期最新研究方向 ........................................................ 5
二、Vertrel XF 於病毒純化的應用 .................................................. 5
三、聚乙二醇（polyethylene glycol, PEG）於病毒純化的應用 ... 6
四、固定化金屬離子親和性管柱 .................................................... 6
（一）純化原理 ........................................................................ 6
（二）金屬離子 ........................................................................ 7
（三）金屬螯合劑 .................................................................... 7
（四）固定相樹酯 .................................................................... 7
（五）利用IMAC 純化病毒顆粒 ............................................ 7
第二章 研究動機 ...................................................................................... 9
第三章 材料方法 .................................................................................... 10
一、細胞與病毒 .............................................................................. 10
（一）IBDV P3009 病毒株 .................................................... 10
（二）DF-1 細胞 (Gallus gallus, chicken embryo fibroblast cell
line) ................................................................................................... 10
（三）DF-1 細胞培養 ............................................................. 10
二、重組蛋白的製備 ....................................................................... 11
（一）VP2-441 次病毒顆粒（subviral particle, SVP）製備 11
（二）VP3（TVP3）重組蛋白製備 ..................................... 12
iv
三、 VP2、VP3 抗體 ..................................................................... 13
四、病毒增殖條件測試 .................................................................. 13
五、病毒力價測定 .......................................................................... 13
六、病毒大量增殖 .......................................................................... 14
七、病毒沈降條件測試 .................................................................. 14
（一）10% PEG 6000 沉澱 .................................................... 14
（二）3.5% PEG 6000 沉澱 ................................................... 15
（三）超高速離心沉降 .......................................................... 15
八、以固定化金屬離子親和性管柱純化病毒 .............................. 15
（一）超高速沈降病毒組 ...................................................... 15
（二）10% PEG 6000 沉澱組 ................................................ 16
九、超高速離心純化病毒 .............................................................. 17
十、純化後病毒的沉降 .................................................................. 17
十一、SDS-聚丙烯硒胺凝膠電泳（SDS-PAGE） ....................... 17
十二、Coomassie blue 染色 ............................................................ 18
十三、硝酸銀染色 .......................................................................... 18
十四、西方墨漬法（Western blot） .............................................. 18
十五、總蛋白濃度定量 .................................................................. 19
十六、總蛋白量、病毒回收率與比感染力的計算 ...................... 19
十七、利用穿透式電子顯微鏡觀察病毒顆粒 .............................. 20
十八、高效能液相層析 .................................................................. 20
第四章 結果 ............................................................................................ 21
一、IBDV 純化流程示意圖 ........................................................... 21
二、IBDV P3009 的增殖條件測試 ................................................ 21
三、IBDV 沉降條件測試 ............................................................... 22
四、以固定化金屬親和性管柱純化病毒 ...................................... 22
（一）超高速離心沉降病毒組 .............................................. 22
（二）10% PEG 6000 沈降病毒組 ........................................ 23
五、以蔗糖梯度離心純化病毒 ...................................................... 24
六、以穿透式電子顯微鏡檢視病毒顆粒 ...................................... 25
（一）傳統方法與IMAC 純度上的差異性 .......................... 25
（二）DF-1 細胞增殖病毒的顆粒多樣性 ............................. 25
v
七、以高效能液相層析管柱分析病毒顆粒 .................................. 25
第五章 討論 ............................................................................................ 27
一、感染條件MOI 的高低與增殖病毒力價的關係 .................... 27
二、不同沉降病毒方法的差異 ...................................................... 27
三、IMAC 與傳統梯度離心純化的結果比較 ............................... 28
四、IMAC 純化IBDV 與VP2 SVP 的差異 ................................. 28
（一）細胞培養基中胎牛血清白蛋白的干擾 ...................... 29
（二）His 253 的暴露程度不同 ............................................ 29
五、不同沉降條件對IMAC 純化結果的影響.............................. 29
六、病毒回收率的增進方法 .......................................................... 30
七、IBDV 於DF-1 細胞增殖的多型性 ......................................... 30
八、45 nm 與60 nm 病毒顆粒的分離 ........................................... 31
（一）膠體過濾層析法（gel filtration） .............................. 31
（二）超高速梯度離心 .......................................................... 31
九、病毒純化的後續應用 .............................................................. 31
（一）病毒結構的解析 .......................................................... 31
（二）病毒感染機制的研究 .................................................. 32
第六章 參考文獻 .................................................................................... 33
表一、病毒沉降條件測試比較 ...................................................... 41
表二、固定化金屬親和性管柱純化經超高速離心沉降之IBDV 結
果分析 ...................................................................................................... 42
表三、固定化金屬親和性管柱純化經10% PEG 6000 沉降的IBDV
結果分析 .................................................................................................. 43
表四、蔗糖超高速離心經PEG 沉降之IBDV 的結果分析 ......... 44

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