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研究生:陳雅薇
研究生(外文):Chen Yai Wei
論文名稱:在昆蟲細胞中所表現傳染性華氏囊病毒結構蛋白的純化及特性分析
論文名稱(外文):Purification and Characterization of the IBDV rVP2H protein expressed in the baculovirus/insect cell expression system
指導教授:王敏盈
指導教授(外文):Wang Mei Yi
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:83
中文關鍵詞:傳染性華氏囊病病毒重組桿狀病毒感染昆蟲細胞似病毒粒子
外文關鍵詞:infectious bursal disease virusinsect cell culture / baculovirus expression systemvirus like particle
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中文摘要
傳染性華氏囊病病毒 (infectious bursal disease virus;簡稱IBDV)是 nonenvelope且有二十面 (icosahedral)對偁的雙股 RNA病毒。其中 VP2及VP3是主要的病毒結構蛋白; VP2是包覆在病毒顆粒外殼的結構蛋白,帶有抗原決定位置,能引發宿主的抗體免疫反應。
本研究是用重組桿狀病毒感染昆蟲細胞Hi-5 ( insect cell culture / baculovirus expression system )來表現 C端接有六個組氨酸之 IBDV病毒中的結構蛋白 rVP2H;之前研究發現結構蛋白 rVP2H會自行組裝成似病毒粒子 (簡稱 VLP),且這些似病毒粒子也能引發中和抗體反應。本研究使用高效能液相層析法HPLC來測定這些由結構蛋白 rVP2H所組裝成的似病毒粒子之熱穩定性;結果發現該蛋白所組裝成的似病毒粒子的熱穩定性和自然界存在的傳染性華氏囊病毒粒子一樣,都可穩定的存在於60℃。但在昆蟲表現系統中也有沒被組裝成似病毒粒子而以其它形式存在的 rVP2H蛋白部分;為了能進一步去分析及純化這些以其它形式存在的 rVP2H蛋白部分,本研究先把所得細胞液以二段式純化方式先把其它形式存在的rVP2H蛋白和似病毒粒子分開:固定化金屬離子親和性分析法 (IMAC)及膠過濾層析法 (CL-6B)處理之後,用 ELISA鑑定結果發現有兩種不同分子量的 rVP2H蛋白存在。本研究為了更進一步了解及純化這些沒有形成似病毒粒子的 rVP2H蛋白是以何種形式存在,選擇凝膠過濾分析法Sephacryl S-200分析其分子量;分析結果發現這些沒有形成似病毒粒子的 rVP2H蛋白是以單體形式 (分子量約為43 kDa)存在。因為在細胞生產 rVP2H蛋白時,大部分都會組裝成似病毒粒子;以此種單體形式存在的 rVP2H蛋白並不多。本研究用 100 kDa的膜來分離似病毒粒子蛋白及單體 rVP2H蛋白後,再用 30 kDa膜來濃縮單體 rVP2H蛋白後;以強陰離子交換樹脂來進一步純化單體 rVP2H蛋白。用此種超濾膜分離程序可達到分離似病毒粒子及單體 rVP2H蛋白之效果。
在蛋白純化工程方面,本研究已找到用固定化金屬離子親和性分析法 (IMAC)純化 rVP2H蛋白的最適化條件,再配合超濾膜分離程序;似病毒粒子蛋白產量已可提升到 250 ml 的細胞培養液可生產出 2 mg的似病毒粒子及 0.5 mg的單體 rVP2H蛋白。未來可再進一步去測試單體 rVP2H蛋白所能引發的免疫反應。
The purification and characterization of different VP2 conformation from virus like particle that formed by a structural protein (VP2) of infectious bursal disease virus (IBDV) was investigated in this study. Besides this, we used high performance liquid chromatography to test the heat stability of virus like particle. Infectious bursal disease virus (IBDV), a number of the Birnaviridase family, is a pathogen of major economic importance to the world''s poultry industries. A gene encoding a structural protein (VP2) of a local strain (P3009) of infectious bursal disease virus (IBDV) was cloned and expressed using the baculovirus expression system to develop a subunit vaccine against IBDV infection in Taiwan. To facilitate the purification of the particles, the VP2 protein was engineered to incorporate a metal ion binding site (His)(6 )at its C-terminus. The chimeric rVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni2+). After immobilized-metal affinity chromatography and gel filtration purification process, we found that there have high molecular weight of rVP2H (VLP) and low molecular weight of rVP2H. In order to find out that the different form of low molecular, we used the Sephacryl S-200 to determine the molecular weight of the different rVP2H conformation. The result was that the different conformation of rVP2H is monomer. In order to get more rVP2H protein we designed a new ultrafiltration purification technique to purify more mono form rVP2H protein and we also have large quantities of rVP2H protein of virus like particle and monomer.
目錄
頁次
中文摘要……………………………………………………….I
英文摘要……………………………………………………….III
目錄…………………………………………………………….V
圖目錄………………………………………………………….
表目錄…………………………………………………………..
第一章 前言……………………………………………………1
第二章 傳染性華氏囊病病毒簡介…………………………….4
2.1 傳染性華氏囊病簡介………………………………….4
第三章 蛋白質濃縮與膜分離技術……………………………..6
3.1蛋白質濃縮與膜分離技術……………………………..6
3.1.1 超微膜濃縮裝置…………………………………..6
3.1.2膜過濾技術…………………………………………7
3.1.3其它濃縮方法………………………………………8
第四章 色層分析法(膠體過濾法,陰離子交換樹脂及固定化金屬離子層析法)………………………………………………………………9
4.1色層分析技術之原理……………………………………………9
4.1.1 膠體過濾法……………………………………………...10
4.1.2 膠體介質………………………………………………...10
4.1.3 膠球大小………………………………………………...11
4.1.4 膠体的選擇…………………………………………… 12
4.2離子交換法…………………………………………………… 12
4.2.1 選擇交換介質…………………………………………..13
4.3 用固定化金屬親和性層析法 (IMAC)來純化 IBDV結構蛋白 rVP2H……………………………………………………………….14
4.4管柱系統………………………………………………………15
4.5 HPLC 與 FPLC系統…………………………………………15
4.6 用液相層析法 (liquid chromatography)純化蛋白過程的探討與產率預測……………………………………………………………17
第五章 在昆蟲細胞中表現之傳染性華氏囊病毒結構蛋白rVP2H
之純化與特性分析研究的材料與方法…………………………….19
5.1病毒株…………………………………………………………19
5.2 細胞培養及培養基……………………………………………19
5.3使用固定化金屬親和性色層分析法純化……………….……19
5.4用凝膠過濾色層分析法純化………………………………….21
5.5用凝膠過濾色層分析法Sephacryl S-200 HR去分析經CL-6B所得之rVP2H…………………………………………………………23
5.6使用陰離交換樹脂 (Anion-exchange column)分析法純化單體的rVP2H部分…………………………………………………………24
5.7用 HPLC來測試似病毒粒子的熱穩定性…………………….26
5.8酵素連結免疫吸附測定 (ELISA)……………………………...27
5.9 MicroplateBCA定量…………………………………………….27
5.10 銀染 (Siliver stain)…………………………………………….28
5.11 凝膠電泳 (Sodium Dodecyl Sulfate-Polyacrylamide )………..28
5.12西方墨點法 (Western blotting)……………………………...29
第六章 結果與討論…………………………………………31
6.1以固定化金屬親和性層析法初純化rVP2H及VLP…………31
6.2以凝膠過濾色層分析法-6B填充管柱分離經IMAC純化所得之VLPs…………………………………………………………………31
6.3以凝膠過濾色層分析法Sephacryl S-200 HR去分析經CL-6B所得之rVP2H…………………………………………………………34
6.4以 Resource Q純化的結果……………………………………35
6.5以 HPLC分析VLP熱穩定性的結果…………………………35
6.6估計似病毒粒子 (VLP)分子量的部分………………………...36
6.7似病毒粒子的特性分析方面……………………………………37
6.8在單體蛋白 ( monomer )特性方面……………………………..37
6.9在純化單體蛋白 ( monomer )部分……………………………..38
6.10蛋白純化方面目前最大單位處理量…………………………..38
第七章結論…………………………………………………………39
參考文獻………………………………………………………………..40
附錄…………………………………………………………..…
參考文獻
何靜宜,1998。利用桿狀病毒表現華氏囊病病毒之大段基因。國立中興大學農業生物科技學研究所碩士論文。
蔡岳華,2000。固定化金屬與 Heparin親和薄膜應用於板框型薄膜分析分離器之設計與研究。國立中興大學化學工程研究所碩士論文。
鄭宇伸,2000。二段式色層分析程序應用於傳染性華氏囊病毒似病毒粒子純化分離之程序設計及研究。國立中興大學生物化學研究所碩士論文
陳敬和,2001。酵母菌轉錄因子 a1蛋白質的純化與量產。國立中興大學生物化學研究所碩士論文
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