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研究生:鍾曜吉
研究生(外文):Yao-Chi Chung
論文名稱:利用昆蟲細胞表現家禽里奧病毒σC蛋白質並以固定化金屬親和層析法純化之研究
指導教授:胡育誠胡育誠引用關係
指導教授(外文):Yu-Chen Hu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
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:70
中文關鍵詞:昆蟲細胞家禽里奧病毒桿狀病毒固定化金屬親和層析法
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家禽里奧病毒(Avian reovirus;ARV)能夠引發數種雞隻疾病,並可能導致感染雞隻死亡。σC蛋白質是家情里奧病毒外層結構蛋白質之一,它和病毒融合入細胞有一定的關係,也是引發中和抗體的主要抗原,因此此病毒就成為我們發展疫苗及檢測試劑的最佳選擇。在這個研究中,我們利用桿狀病毒/昆蟲細胞表現系統表現σC蛋白質並研究觀察其表現特性。我們在此實驗中表現的σC蛋白質產量,每106細胞能高達96 mg,相對於其他種類的病毒蛋白質在此種表現系統中的產量,我們的表現量是相當高的。此外,我們將σC蛋白質融合了額外增加的histidine tag及EK切位,融合這些氨基酸將有助於蛋白質表現後的純化,以及接下來的去除histidine tag的工作。我們成功地利用固定化金屬親和層析法以有效的單一步驟純化,能夠在一步驟中得到純度高達94%的產物,而且其回收率約為58%。接下來,我們又成功地以酵素enterokinase(EK)將純化後蛋白質的hitidine tag除去。總而言之,我們將σC蛋白質融合histidine tag及EK切位後的融合蛋白,能夠在桿狀病毒/昆蟲細胞表現系統中大量表現;在純化步驟中,此重組蛋白質上的histidine tag能和IMAC上的鎳離子(Ni2+)結合而純化,EK切位也能有預期的效果,因此這個成功的融合策略使我們能以成功而簡易的方法達到純化的目的。

Avian reovirus (ARV) causes a number of severe chicken diseases and death. One of the outer structural proteins, sC, is linked to cell fusion and neutralizing antibody induction, and therefore becomes the prime candidate for vaccine development and diagnosis tool. In this study, we expressed sC using baculovirus/insect cell system and characterized the production profiles. The yield reached 96 mg/106 cells, which is high compared to other viral proteins expressed in this system. Besides, the protein was fused with additional histidine tag and an EK cleavage site to facilitate the downstream purification and subsequent removal of the histidine tag. Successful and efficient one-step purification using immobilized metal affinity chromatography (IMAC) was demonstrated and the recovery was roughly 58%. The purity by a single step reached 94%. Additionally, the histidine tag was successfully removed by incubating the purified protein with EK. Overall, the fusion strategy resulted in high level expression of sC properly fused to a histidine tag and an EK cleavage site, the recombinant protein conformation conferred reasonable accessibility of the histidine tag to Ni2+ for IMAC purification and the enterokinase cleavage, and enabled successful, simple downstream purification.

目錄………………………………………………………………………I
圖表目錄………………………………………………………………...II
第一章 緒論
1-1 前言…………………………………………………………………1
1-2 研究動機……………………………………………………………3
第二章 文獻回顧
2-1 家禽里奧病毒的簡介……………………………………………5
2-2 桿狀病毒表現系統簡介………………………………………9
2-3 固定化金屬親和層析法原理簡介………………………………13
第三章 實驗材料及方法
3-1 材料………………………………………………………..………16
3-2 實驗方法………………………………………..………………18
第四章 結果與討論
4-1 Avian reovirus σC基因轉殖載體之製備…………....…41
4-2 重組病毒之製備…………………………………………45
4-3 Avian reovirus S1133 σC蛋白質的生產…………..…..47
4-4 以IMAC純化重組蛋白σC-His……………………….54
第五章 結論…………………………………………………...62
參考文獻………………………………………………………………64
附錄…………………………………………………………………67
圖表目錄:
圖2-1 家禽里奧病毒的外觀簡圖……………………………………....6
表2-1 家禽里奧病毒所有基因、表現蛋白質及蛋白質位置……..….7
圖2-2 家禽里奧病毒的蛋白質分佈概略圖……………………………7
圖2-3 病毒和轉殖載體間的同源互換………………………………..12
圖2-4 蛋白質額外添加6個組氨酸以利於純化……………….…….15
表3-1 微量質體DNA萃取法各溶液成分…………………………….19
表3-2 PCR反應的配方………………………………………………...21
表3-3 PCR反應的時間………………………………………………..21
表3-4 繼代培養時不同培養容器所需的要放入的細胞量及培養液總體積或濃度……………………………………………….…………….24
圖3-1 病毒藍點形成過程…..…………………………………………27
表3-5 SDS-PAGE所使用的膠體配方…………………………………32
表3-6 對不同蛋白質所使用的第一及第二抗體……………………..33
表3-7 各種緩衝液的配方及濃度……………………………………..34
表4-1 班點分析法不同的稀釋倍率下出現之藍點………………….44
表4-2 貼附培養及懸浮培養在不同時間時不同病毒感染狀況下的存活率(%)………………………………………………………………45
表4-3:各個純化步驟的損失和產率………………………………….51
圖4-1 轉殖載體製作流程圖…………………………………………..54
圖4-2 以凝膠電泳分析經限切酵素處理過後的σC基因及pBlueBacHis-2. …………………………………………………...……55
圖4-3經抗生素篩選後的16株菌的載體以限切酵素Kpn I及Hind III作用…………………………………………………………………….55
圖4-4 共轉染後的菌斑……………………………………………….56
圖4-5 貼附培養及懸浮培養在不同時間時不同病毒感染狀況下的存活率(%)………………………………………………………………..57
圖4-6 以SDS-PAGE分析蛋白表現之最佳條件及時間點圖………..58
圖4-7 以Western Blot分析感染後細胞內所生產的蛋白質………....59
圖4-8 重組病毒感染3天後蛋白質分佈…...…………………………59
圖4-9 在純化過程中不同樣本所對應的吸收值…………………..…60
圖4-10 純化過程的SDS-PAGE分析………………………………...60
圖4-11 純化過程以西方點墨法分析……………………………...….61
圖4-12 Enterokinase對denature及native的σC-His蛋白質的作用..61
圖4-13 EK的作用(a)SDS-PAGE;(b)anti-ARV抗體的Western-Blot;(c)anti-His抗體的Western-Blot…………………………………….62

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