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研究生:蔡倩妞
研究生(外文):Chian New Chua
論文名稱:傳染性雞華氏囊病毒的VP2蛋白於大腸桿菌中組裝成次微米級似病毒顆粒之研究
論文名稱(外文):Self-assembly of nano-particle by infectious bursal disease virus VP2 structural protein in Escherichia coli
指導教授:王敏盈
指導教授(外文):Min-Ying Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:76
中文關鍵詞:傳染性雞華氏囊病毒似病毒顆粒結構蛋白
外文關鍵詞:infectious bursal disease virusvirus-like particlestructural protein
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本研究是將傳染性雞華氏囊病毒的結構蛋白VP2基因構築於表現載體中,利用大腸桿菌表現VP2及C端截切的VP2蛋白,經由不同條件測試,探討其是否能自我組裝形成似病毒顆粒(virus-like particle, VLP)。大腸桿菌表現未帶有任何融合蛋白的全長erVP2可溶蛋白,經氯化銫密度梯度離心,於電子顯微鏡下觀察可見粒徑大小約為20 nm次微米級似病毒顆粒。然而在C端融合了六個組胺酸(6 histidine residues)的erVP2H的可溶蛋白在大腸桿菌BL21(DE3) CodonPlus-RP表現量不高,雖然可以藉由固定化金屬離子色層分析法純化,在電子顯微鏡的觀察下其所形成的為15 nm似病毒粒子。VP2蛋白在大腸桿菌中形成似病毒顆粒的研究未曾被報導,因此本實驗繼續利用此系統表現另外兩個C端截切的VP2蛋白─er1167H及er1197H,並於C端也融合了六個組胺酸,但這兩個不同長度C端截切及融合六個組胺酸的VP2蛋白則未見有似病毒顆粒的形成。另一方面,erVP2蛋白已藉由發酵槽來進行大規模生產。本研究所探討的erVP2與erVP2H蛋白皆能組裝形成特定的結構,這是在以往的研究當中從未發現的現象,因此相信這兩種特定結構在雞隻的免疫過程中能誘發雞隻產生很強的免疫反應。
Abstract
Gene encoding a structural protein (VP2) of infectious bursal disease virus (IBDV) was cloned and expressed using the host, Escherichia coli (E. coli), to investigate its capability of the formation of particulate structure. Although the previous attempt to produce unfused VP2 in E. coli was reported to be unsuccessful, in this report, the soluble E. coli-derived rVP2 (erVP2) proteins were found to form particles of approximately 20 nm in diameter. Those particles were partially purified employing CsCl density gradient ultracentrifugation, and confirmed by direct observation under the electron microscope. To facilitate the purification of the particles, the VP2 protein was incorporated a metal ion binding site (His)6 at its C-terminus. The chimeric erVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni+2). However, the chimeric erVP2H particles have a slightly smaller particulate size, approximately, 15 nm. The solubility of erVP2H is extremely low in the strain of BL21(DE3)pLysS and it was expressed with a limited amount in the strain of BL21(DE3)CodonPlus-RP. Besides, two C terminal deletion mutant proteins of VP2, 1167H and 1197H protein also failed to form particles. Thus, the erVP2 protein was selected to express in a laboratory-scale fermentor. In conclusion, the most significant finding in this work is that both of the expressed erVP2 and erVP2H proteins can form a particulate structure, which is not found previously and is believed to induce a strong immunological response in a vaccinated chicken.
中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………2
一、前言………………..………………………………………………………………3
二、文獻回顧…………..………………………………………………………………5
2-1 傳染性雞華氏囊病之簡介……………………………………………………….5
2-2 傳染性雞華氏囊病毒…………………………………………………………….5
2-2-1 IBDV之分類…………………………………………………………………5
2-2-2 IBDV之理化特性……………………………………………………………6
2-2-3 IBDV的基因組及其蛋白組成………………………………………………6
2-2-4 IBDV之增殖和致病機轉……………………………………………………8
2-3 IBDV疫苗的發展………...………………………………………………………8
2-3-1 IBDV次單位疫苗的研究─大腸桿菌與酵母菌表現系統…………………8
2-3-2 IBDV次單位疫苗的研究─昆蟲細胞/桿狀病毒表現系統……...…..……11
2-3-3 IBDV次單位疫苗的研究─其他...……………………………….………..11
2-4 大腸桿菌表現外源蛋白的應用………………………………………………...12
三、材料與方法…………………………………………………………..………..…15
3-1 引子的設計..…………………………………...……………………………….…..15
3-2 聚合酵素連鎖反應(PCR)之反應條件…………………………………………15
3-3 產物的確認……………………………………………………………………...16
3-4 接合作用(ligation)……………………………………………………………..16
3-5 重組質體之轉型作用(transformation)…………………………….…………..17
3-6 小量質體的製備…………………………………………………….…………..17
3-7 重組質體的純化…………………………………………………….…..………17
3-8 VP2蛋白的表現……………………………………………………....…………18
3-9 生物反應器-發酵槽之培養方式.………………………………………………19
3-10 VP2蛋白之純化與分析………………………………………….………….…19
3-10-1 SDS polyacrylamide gel electrophoresis(SDS-PAGE)…….……………..19
3-10-2 硝酸銀染色法(silver staining)…………………………………………...20
3-10-3 西方墨點法(Western blotting)…………………………………..……….20
3-10-4 固定化金屬親和性色層分析法─Immobilized metal-ion affinity chromatography(IMAC) …………………………………………………..21
3-10-5 氯化銫密度梯度離心…………………………………………………….21
3-10-6 酵素連結免疫分析法(Enzyme-linked immunoassay, ELISA)………..22
3-10-7 凝膠過濾色層層析法(HPLC-based gel filtration chromatography).……22
3-10-8 穿透式電子顯微鏡觀察法(Transmission electron microscopy, TEM)23
3-10-9 似病毒顆粒之免疫金粒子的標定(immunogold labeling).……………...23
3-11 雞隻免疫實驗………………………………………………………………….24
3-11-1 雞胚胎纖維芽細胞(chicken embryo fibroblast, CEF)之製作…………..24
3-11-2 病毒中和實驗(virus neutralizing assay)………………………………....24
四、結果與討論………………………………………………...…….………………26
4-1 質體的構築…………………………………………………………….………..26
4-2 er1167H蛋白的研究分析……………………………………………………….27
4-2-1 er1167H蛋白的表現………………………………………………………..27
4-2-2 包涵體型式er1167H蛋白的純化及抗體的製備………………………....27
4-2-3 可溶er1167H蛋白的純化……………………………………..…………...28
4-2-4 er1167H蛋白之穿透式電子顯微鏡觀察…………………………………..28
4-3 er1197H蛋白的研究分析…………………………………………………….…28
4-3-1 er1197H蛋白之表現、純化與分析…………………………………………28
4-4 erVP2H蛋白的研究分析……………………………………………………..…29
4-4-1 erVP2H蛋白的表現、純化及分析………………………………………….29
4-5 erVP2蛋白的研究分析……………………………………………..…………...31
4-5-1 生物反應器─發酵槽的最適化培養……………………………………...32
4-5-2 氯化銫密度梯度離心分離及純化erVP2蛋白……………………………33
4-5-3 以凝膠過濾色層層析法及ELISA分析鑑定erVP2似病毒顆粒…………34
4-5-4 以Triton-X-100處理超高速離心後的erVP2似病毒顆粒………………..34
4-5-5 以凝膠過濾色層層析法及ELISA分析鑑定經由Triton-X-100處理後
的erVP2似病毒顆粒…………………………………………………...…35
4-5-6 免疫金粒子的標定……………………………………………...…………36
4-6 雞隻免疫實驗…………………………………………………………………...36
五、討論………………………………………………………………………………37
六、結論……………………………………………………………………………....40
七、參考文獻……………………………………………………..…………………..41
圖表…………………………………………………………………………………..52
附錄…………………………………………………………………………………..75
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