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研究生:林雋倫
研究生(外文):Chun-Lun Lin
論文名稱:結合超過濾系統與固定化金屬親合力層析法以純化昆蟲桿狀病毒
論文名稱(外文):Combination of Ultrafiltration and Immobilized Metal Affinity Chromatography for the Purification of Baculovirus
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:2004
畢業學年度:92
語文別:中文
論文頁數:53
中文關鍵詞:昆蟲桿狀病毒固定化金屬親合力層析法病毒純化基因治療
外文關鍵詞:baculovirusimmobilized metal affinity chromatography (IMAC)virus purificationgene delivery
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:1
昆蟲桿狀病毒(baculovirus)已於1995年證實可感染哺乳動物細胞,因此被認為具有成為基因治療載體的潛力。若要將其應用在基因治療載體方面,先決條件是取得高純度及高濃度之桿狀病毒。傳統的多次超高速離心純化法因相當費時費力且較難以規模放大,並不適合作病毒載體的大量純化。相對地,層析法已廣泛應用於蛋白質純化 但尚未普遍應用於基因治療載體之大規模純化。因此,本實驗室先前應用固定化金屬親合力層析法(IMAC)結合超過濾(ultrafiltration)系統純化桿狀病毒,在IMAC部分回收率可達8 %。為提升回收率以使此結果可以應用至大規模純化,在本研究中針對IMAC及超過濾系統的各項參數予以改進,這些參數包含IMAC進料流速(提升病毒結合速率並縮短操作時間),IMAC溶液鹽度(提升病毒結合速率並減低對病毒傷害),IMAC溶液pH值(提升病毒結合速率並減低對病毒傷害),IMAC沖提液梯度(提升純化效率),超過濾濾膜孔徑(提升前處理效率)以提升回收率及純度等。並以終點稀釋法(EPDA, End Point Dilution Assay)及流式細胞儀作為監測每一純化步驟病毒回收率的工具,希望能找出適當的純化步驟。
Baculovirus has been proved to be able to infect mammalian cells at 1995 and have the potential as a gene delivery vehicle. As a gene therapy vector, generation of high viral purity and concentration is prerequisite. Traditional virus purification by several ultra centrifugations is not suitable for virus purification on large scale, because it is time consuming and hard to scale up. Comparatively, chromatography has been widely used on protein purification but has not been widely used on mass purification of gene therapy vehicle. In precious research of our laboratory, we combine immobilized metal affinity chromatography (IMAC) and ultrafiltration system to purify baculovirus, and the recovery of virus titer is 8 % in IMAC process. To apply this result for purification on large scale, we should improve virus recovery. In this research, we try to modify parameters of IMAC and ultrafiltration, such as feed flow rate of IMAC (to improve virus combining rate and shorten operating time), salt concentration of IMAC buffers (to improve virus combining rate and shorten operating time), pH of IMAC buffers (to improve virus combining rate and shorten operating time), gradient of IMAC elution buffer (to improve purification efficiency), and pore size of ultrafiltration membrane (improve ultrafiltration efficiency) to improve recovery and purity. We try to find applicable purification process by using End Point Dilution Assay (EPDA) and flow cytometry to detect virus recovery in each step of purification process.
目 錄
第一章 文獻回顧..........................................1
1-1 昆蟲桿狀病毒簡介.....................................1
1-2 桿狀病毒應用於基因治療之潛力.........................1
1-3 超過濾系統...........................................2
1-4 固定化金屬親和力層析法...............................3
1-5 研究動機.............................................3
第二章 實驗材料與方法....................................8
2-1 昆蟲細胞培養.........................................8
2-2 哺乳動物細胞(BHK)培養................................8
2-3 放大桿狀病毒.........................................8
2-4 以流式細胞儀計數病毒顆粒.............................9
2-5 以終點稀釋法計算病毒濃度 ...........................10
2-6 以流式細胞儀計算病毒感染哺乳動物細胞效率............10
2-7 以超過濾系統濃縮病毒................................10
2-8 以IMAC純化經超過濾系統濃縮後的病毒..................11
第三章 實驗架構.........................................14
第四章 實驗結果及討論......................................................16
4-1 提升病毒生產效率....................................16
4-2 自然接合液的pH值對病毒活性的影響....................17
4-3 自然接合液的鹽類濃度對病毒活性的影響................19
4-4 Bac-CEH病毒結構的效應...............................21
4-5 以哺乳動物細胞評估病毒活性的變化....................22
4-6 在自然接合液中溶液中加入Ca2+, Mg2+的效應............24
4-7 超過濾系統中轉速的影響..............................26
4-8 超過濾系統中濾膜的孔徑大小與操作轉速對病毒回收率的共
同效應..............................................27
4-9 超過濾系統緩衝液交換策略............................29
4-10 IMAC病毒灌流方式對結合效率的影響...................31
第五章 結論.............................................44
第六章 未來努力方向.....................................46
參考文獻................................................48

圖表目錄
圖1-1 桿狀病毒的分類.....................................5
圖1-2 各種濃縮方法的使用範圍比較.........................5
圖1-3 組胺酸的共振結構以及形成的錯合結構.................6
圖1-4 固定化金屬親合力層析法(IMAC)的基本原理...........6
表1-1 IMAC與其他管柱層析法的優劣比較.....................7
圖2-1 Stirred Cell裝置圖................................12
表2-1 在IMAC純化中所使用的緩衝溶液配方..................13
圖3-1 本研究主要架構....................................15
圖4-1 溶液pH值對病毒活性及顆粒數的影響..................33
表4-1 溶液pH值對病毒影響:粒子與感染力比值及保存率......33
圖4-2 溶液鹽度對病毒活性及顆粒數影響....................34
表4-2 溶液鹽度對病毒影響:粒子與感染力比值及保存率......34
表4-3 以滲透壓儀測定2-7,2-8節中使用溶液的滲透度........35
表4-4 2-7,2-8節中使用溶液的滲透度與病毒生產時滲透度差異35
圖4-3 不同病毒在PBS及自然接合液中其活性及顆粒數的變化...36
圖4-4 不同病毒量與病毒對BHK感染力的關係.................37
圖4-5 不同病毒經過PBS或自然接合液處理後,其對BHK的感染力變
化................................................37
圖4-6 在自然接合液中,摻入Ca2+及Mg2+對改善病毒活性及對BHK感
染力的幫助........................................38
圖4-7 超過濾系統內轉速對病毒活性及顆粒數之影響..........39
圖4-8 超過濾系統內濾膜孔徑及操作轉速對除去病毒液雜蛋白效率
的影響............................................40
表4-5 濃縮方式對病毒液最終吸光值的影響..................40
圖4-9 超過濾系統內濾膜孔徑與操作轉速對病毒活性,及病毒顆粒
數的影響..........................................41
圖4-10 修正超過濾步驟對除去病毒液雜蛋白效率的影響.......42
表4-6 修正超過濾步驟對超過濾系統中病毒保存率的各項指標的影
響................................................42
圖4-11 灌流次數對病毒與樹脂結合力的變化.................43
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