家禽流行性感冒病毒對世界家禽產業造成難以估計的經濟損失，而其所衍生出的鳥傳人，豬傳人，及人傳人的變種，其高至死率使我們迫切的需要發展疫苗以控制疫情。Bac-HA64為一表面帶有禽流感病毒套膜(envelope)的血球凝集素(Hemagglutinin, HA)蛋白於桿狀病毒套膜上之偽型桿狀病毒，並已在本實驗室先前的動物實驗中初步證實其具有引發中和血球凝集素抗體之效用。因此我們希望在疫苗應用上發展一套便利的製程來大量生產及純化此偽型桿狀病毒Bac-HA 64。在量產方面，我們結合國內賽宇細胞科技公司所開發出的新式拋棄式填充床反應器BelloCellÒ，可有效大量培養細胞，且具有低剪應力之優點，預期可減低桿狀病毒在生產過程之傷害，所以我們將其用來生產桿狀病毒。首先我們在篩選Sf 900 II、TNM、ESF 921三種培養基後，發現Sf 900 II可產出最高之病毒效價，此外BelloCellÒ相較於旋轉瓶，在產Bac-HA64上雖無明顯提升，但生產Bac-CHA時，效價則有近三倍之提升。在純化方面，先前實驗室已成功建立利用切向流過濾（tangential flow filtration, TFF）用以濃縮並將病毒環境溶液置換為緩衝液，我們在此將再結合膠體過濾層析法來純化桿狀病毒。TFF是個很省時且易於製程放大的超過濾系統，除了濃縮外，尚可以移除部份較小的雜蛋白，回收率約在65%；而膠體過濾層析純化法是利用分子大小不同，造成通過管柱時間不同來分離其他雜質，回收率約在65.5 %，耗時約50 min。整個純化流程回收率達42.2 %，純度方面達到70%以上。本實驗中所建立的生產程序，在效價上有著跟傳統的旋轉瓶一樣甚至更好的效果，但卻是種非常容易放大製程的生產方式。純化程序回收率遠比傳統的超高速離心(<1%)來得高，且是種非常省時且容易大量的純化程序。

目錄 IV
圖目錄 VI
表目錄 VI
第一章 緒論 1
1-1家禽流行性感冒病毒及其疫苗 1
1-2昆蟲桿狀病毒/昆蟲細胞表現系統 2
1-3血球凝集素於昆蟲桿狀病毒表面上之修飾 3
1-4生物反應器 4
1-4-1 生物反應器 4
1-4-2 BelloCell® 5
1-5超微膜過濾濃縮技術 6
1-6膠體過濾管柱層析法 8
1-6-1膠體過濾層析 8
1-6-2 Sephacryl S-300、S-400 High Resolution 9
1-7研究動機 9
第二章 實驗材料與方法 18
2-1細胞培養 20
2-1-1昆蟲細胞培養 20
2-1-2哺乳動物細胞培養 20
2-2重組昆蟲桿狀病毒之放大 20
2-3新型生化反應器BelloCell 500® 21
2-3-1反應器構造 21
2-3-2接種 22
2-3-3取樣 22
2-3-4換液 22
2-3-5感染病毒 22
2-4桿狀病毒在不同層析溶液的轉導能力 23
2-5切向流超過濾濃縮系統（tangential flow filtration，TFF） 23
2-6利用膠體過濾層析法純化桿狀病毒 24
2-7分析儀器與方法 25
2-7-1 Crystal violet dye (CVD) method 25
2-7-2 GlucCellTM glucose monitoring system 25
2-7-3終點稀釋法測定病毒感染效價 25
2-7-4以流式細胞儀（flow cytometry）測定並計算病毒轉導效價 25
2-7-5 SDS-PAGE 27
2-7-6西方點墨法（Western-blot） 27
2-7-7 NOVA (Nova BioProfile 100 analyzer) 28
2-8統計分析 28
第三章 結果與討論 29
3-1重組桿狀病毒生產程序的改善 29
3-1-1昆蟲細胞株之探討 29
3-1-2培養基之探討 29
3-2 BelloCell 500□生產重組桿狀病毒最適化條件探討 30
3-2-1 BelloCell內細胞生長條件最適化 30
3-2-2感染時機與MOI之探討 30
3-2-3病毒表面帶有HA對效價之影響 32
3-3純化程序之改善 33
3-3-1不同buffer對病毒穩定性之影響 33
3-3-2切向流過濾膜的孔徑大小對回收率之影響 34
3-3-3流速、膠體孔徑及純化後回收率及純度之探討 35
第四章 未來展望 50
4-1生產重組桿狀病毒最適化條件探討 50
4-1-1 壓縮速率的探討 50
4-1-2 停滯時間之探討 50
4-2 SEC膠體孔徑大小最適化 51
參考文獻 52

圖目錄
圖 1- 1昆蟲桿狀病毒粒子AcMNPV的穿透式電子顯微鏡照片。 11
圖 1- 2昆蟲桿狀病毒的感染與複製週期。 12
圖 1- 3 一般病毒載體的生產及純化流程示意圖。 13
圖 1- 4 (a)BelloCell□500 反應器及BelloStage 3000示意圖 (b)BelloCell運作原理 (c)BelloCell P運作示意圖。 14
圖 1- 5樣本體積與不同超過濾系統關係圖。 15
圖 1- 6切向流過濾原理示意圖。 16
圖 1- 7濾膜與分子大小對照圖 17

圖 3- 1 不同細胞株對病毒產量的影響。 38
圖 3- 2不同培養基對病毒的影響。 39
圖 3- 3用Sf 900 II培養之Sf-9細胞於BelloCell 500內之生長曲線圖。 40
圖 3- 4 以BelloCell反應器搭配不同細胞數及MOI組合對病毒效價之影響 41
圖 3- 5表面帶有HA對效價之影響 43
圖 3- 6 各buffer之total FI值 . 44
圖 3- 7 各孔徑之回收率。 45
圖 3- 8 FPLC純化結果。(a)S-400 HR以不同流速沖洗。(b)S-300 HR以不同流速沖洗 . 46
圖 3- 9 以6.3 % C.V.的樣品量打入管柱(S-300)所得到之FPLC圖。 47
圖 3- 10 利用 (b) SDS-PAGE stained by Coomassie Blue分析各生產及純化步驟完的純度。和 (c)Western Blot 確認病毒存在。 48


表目錄
表 2- 1 各項緩衝液條件。 23
表 2- 2 SDS-PAGE膠體組成 27

表 3- 1 利用BelloCell所生產之桿狀病毒在經過TFF及SEC純化後各步驟回收率 . 49

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