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博碩士論文 etd-0908110-160646 詳細資訊
Title page for etd-0908110-160646
論文名稱
Title
修飾外殼蛋白碳端胺基酸序列影響龍膽石斑神經壞死病毒顆粒組 裝之研究
The effects of C-terminus modification of Dragon Grouper Nervous Necrosis Virus capsid protein on the virus particle formation.
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
179
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2010-06-22
繳交日期
Date of Submission
2010-09-08
關鍵字
Keywords
金屬親合層析法、似病毒顆粒
Dragon Grouper Nervous Necrosis Virus, Circular Dichroism, poly-His tagged VLPs
統計
Statistics
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中文摘要
摘要
本研究以大腸桿菌表現系統表現六種碳端不同長度的外殼蛋白,以探討碳端胺基酸如何影響病毒顆粒的組裝,這些截短的外殼蛋白皆在碳端再加長六或三個組胺酸,作為金屬親合性標籤 (metal affinity tag),用于固定化金屬親合層析法 (Immobilized metal affinity chromatography, IMAC) 來純化帶有 His Tag 的外殼蛋白或似病毒顆粒。△C334-C6H 為碳端被截短四個胺基酸再加上六個組胺酸或碳端未截短者加上六個組胺酸 (C6H),兩者皆無法形成似病毒顆粒。而截短三個胺基酸後加上六個組胺酸的 △C335-C6H,其病毒顆粒產率降低44%,並且病毒結構較不穩定。全長蛋白碳端融合三個組胺酸的 C3H,或截短二至三個胺基酸序列並融合六個組胺酸的 △C336-C6H及 △C337-C6H,其似病毒顆粒的產率皆高於野生型。使用圓二色光譜觀察上述似病毒顆粒與氮端融合 His6 Tag 的似病毒顆粒 (N6H VLPs) 的熱變性過程與熱穩定性,其中被修飾的似病毒顆粒其Tm值皆低於野生型似病毒顆粒 (61℃) 約3℃。使用上述似病毒顆粒,利用 poly-His tag 與IMAC或anti-6xHis antibody 的親和力強弱,探討外殼蛋白氮端和碳端外露程度,於西方墨漬法的結果顯示,Anti-6xHis monoclonal antibod 無法辨認 N6H 外殼蛋白,但由於 N6H 似病毒顆粒與 IMAC 親和力低於碳端 poly-His tagged VLPs,因此推測外殼蛋白碳端較氮端外露於病毒顆粒表面上。
Abstract
In order to investigate the effects of C-terminus modification of Dragon Grouper Nervous Necrosis Virus capsid protein on the virus particle formation, we used E. coli expression system to express DGNNV capsid protain with different truncations at C-teminus fused with six or three histidines (His-Tag). These poly-His tagged clones, including △C334-C6H, △C335-C6H, △C336-C6H, △C337-C6H, C3H and C6H (His6 tagged at the C-teminus of wild-type capsid protein),were expressed and VLPs formation ability were examined. Wild-type and N-terminal recombination (N6H, His6 tagged at the N-teminus of wild-type capsid protein) were also used for comparison. These His-tagged VLPs can be further purified by Ni-NTA agarose, and their thermal stability of mutant VLPs were analyzed by Circular Dichroism. The Western blotting and ELISA assay were utilized to analyzed N-teminus or C-terminus was located at the surface of virus icosahedron. Once the four amino acids at the C-terminus of capsid protein were truncated (△C334-C6H), the mutated cpasid protein cannot assemble into VLPs. The same phenomenon was also observed in C6H. The related productions of wild-type, △C335-C6H, △C336-C6H, △C337-C6H, C3H VLPs were about 100%, 56%, 116%, 141%, and 193%, respectively. Using Circular Dichroism to observe the thermal stability of mutant VLPs, the results revealed that the Tm of mutant VLPs were about 3oC lower than wild-type VLPs (61oC). The results of Western blotting and ELISA assay suggest that the C-termius of DGNNV capisid protein was exposed to the surface of virus structure.
目次 Table of Contents
摘要..................................................................................................................................... I
Abstract.............................................................................................................................. ΙΙI
目錄..................................................................................................................................... IV
表目錄................................................................................................................................. VI
圖目錄................................................................................................................................. VII
壹、前言............................................................................................................................... 1
一、魚類神經壞死病毒 (Piscine Nervous Necrosis Virus).......................................... 1
二、病毒性神經壞死症 (Viral Nervous Necrosis) 之臨床表徵................................. 4
三、Betanodavirus之遺傳物質及其基因功能............................................................. 5
四、似病毒顆粒與其應用............................................................................................. 10
五、物理因子及化學條件降低 Betanodavirus 活性................................................. 17
六、Betanodavirus之檢測法......................................................................................... 17
七、增殖 Betanodavirus 之細胞株.............................................................................. 20
八、Betanodavirus 引發魚類細胞株之細胞凋亡....................................................... 23
九、Poly-His tagged外殼蛋白之應用.......................................................................... 25
十、圓二色 (Circular Dichroism, CD) 光譜儀觀測蛋白質結構的變化.................... 28
十一、研究目的............................................................................................................. 30
貳、材料與方法................................................................................................................... 32
一、質體萃取................................................................................................................. 32
二、DNA電泳................................................................................................................ 33
三、膠體中純化DNA.................................................................................................... 33
四、製作勝任細胞 (Competent Cell)........................................................................... 34
五、細胞轉型 (Cell Transformation)............................................................................ 35
六、SDS-PAGE膠體電泳.............................................................................................. 35
七、Poly-His tagged capsid protein選殖株之設計與製備........................................... 37
八、選殖株表現poly-His tagged capsid protein........................................................... 38
九、表現與純化似病毒顆粒......................................................................................... 38
十、連續式分光光度計分析似病毒顆粒..................................................................... 40
十一、Lowry Method定量蛋白質濃度......................................................................... 41
十二、製備抗VLPs之小鼠血清................................................................................... 42
十三、西方墨漬法 (Western Blotting Analysis)........................................................... 42
十四、ELISA (Enzyme Linked Immunosorbent Assay)................................................ 43
十五、使用 Ni-NTA column 純化 poly-His tagged VLPs......................................... 44
十六、使用 Ni-NTA column 純化 18k supernatant poly-His tagged 外殼蛋白…... 45
十七、圓二色光譜測定 VLPs 之熱穩定性................................................................ 46
十八、電子顯微鏡樣品之製備與觀察......................................................................... 46
參、結果............................................................................................................................... 48
一、Poly-His tagged capsid protein 選殖株設計.......................................................... 48
二、選殖株表現病毒外殼蛋白..................................................................................... 48
三、選殖株生產 VLPs 之能力分析............................................................................ 49
四、各選殖株中病毒外殼蛋白組裝 VLPs 之能力分析........................................... 50
五、穿透式電子顯微鏡觀察 VLPs 形態................................................................... 51
六、Poly-His tagged VLPs 吸附於 Ni-NTA agarose 最適氯化鈉濃度.................... 52
七、使用 Ni-NTA column 純化 poly-His tagged VLPs............................................. 53
八、Ni-NTA column純化細胞溶裂液中的poly-His tagged 外殼蛋白.................... 55
九、ELISA 偵測 VLPs 與 mAs_VLP antiserum 結合效力..................................... 57
十、ELISA 偵測 Anti-6xHis monoclonal antibody 與 VLPs 結合效力………….. 57
十一、Western blotting analysis 偵測病毒外殼蛋白與 Anti-6xHis antibody、mAs_VLP antiserum 結合能力 ...................................................................... 59
十二、似病毒顆粒於連續增溫下的熱穩定性............................................................. 59
肆、討論............................................................................................................................... 66
一、外殼蛋白 C 端影響似病毒顆粒的組裝………………………………………... 66
二、E. coli原核表現系統表現 poly-His tagged 外殼蛋白..……………………….. 66
三、poly-His tagged外殼蛋白組裝似病毒顆粒之能力……...……………………… 67
四、利用Ni –NTA column 純化poly-His tagged VLPs 及純化細胞溶裂液中的
poly-His tagged外殼蛋白………………….…………….…………………….... 70
五、DGNNV N端和C端之外露程度……...………………......................................... 75
六、Poly-His tagged VLPs之熱穩定性…………..…...………………………………. 79
伍、參考文獻....................................................................................................................... 81
陸、圖表............................................................................................................................... 97
附錄A................................................................................................................................. 141
附錄B................................................................................................................................. 142
附錄C................................................................................................................................. 143
附錄D................................................................................................................................. 144
附錄E................................................................................................................................. 145
附錄F................................................................................................................................. 146
附錄G................................................................................................................................. 147
附錄H................................................................................................................................. 168

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