臺灣博碩士論文加值系統

摘要

隨著生化科技的進步，人類發展了許多外源蛋白生產系統，並運用其生產高價值之醫療蛋白，這些系統包含大腸桿菌系統、植物細胞系統、哺乳動物細胞系統及桿狀病毒－昆蟲細胞系統，這些系統各有其利弊，大腸桿菌系統成本最低廉，但卻無法生產需要複雜修飾之外源蛋白：而植物細胞系統雖然可以生產類似真核細胞修飾之外源蛋白，但其修飾作用與人類尚有些許不同，而對於外源蛋白的修飾與人類最接近的哺乳動物細胞系統，卻有成本高及產量偏低等缺點；為解決此一問題在1983年史密斯等人開發出桿狀病毒－昆蟲細胞系統，此一系統有提高外源蛋白產量的優點，但由於昆蟲細胞為桿狀病毒天生之宿主,故在細胞受病毒感染後，細胞將會產生胞裂而無法持續生產外源蛋白；此外雖然桿狀病毒-昆蟲細胞系統具有較植物系統與微生系統較佳之外源蛋白修飾作用但仍缺乏缺乏N-醣基化修飾作用，因此無法生產需醣基化修飾之外源蛋白。而為了改善桿狀病毒－昆蟲細胞之缺陷，本研究將以哺乳類動物細胞取代昆蟲細胞來架構桿狀病毒－哺乳類動物細胞系統，由於桿狀病毒－哺乳類動物細胞系統，需高力價病毒轉染才能具有較高之外源蛋白產量，因此本研究將針對提高病毒濃度及病毒轉染之方式進行探討，藉以提高外源蛋白產量。

關鍵字：昆蟲細胞、桿狀病毒、哺乳類動物細胞

Abstract


Recently, many systems have been developed for the production of protein with medical significance. The baculovirus expression vector system is one of the best expression systems for high yield protein production in insect and mammalian cells with proper posttranslational modifications. In these experiments, baculovirus was produced in insect cell for subsequent protein production in mammalian cells. The best viral yield was achieved by virus infection at the third day and harvesting at 6 days post viral infection. Feed-batch culture yielded roughly twice viruses(ca. 3.8×108 pfu/ml) compare with that produced by batch culture. Optimal viral infection conditions for protein in mammalian cells also explored using β-galactosidase as reporter and found that a 4 fold better yields were achieved by multiple(ca. 1.265mg/L) than by single baculovirus transduction. This experiment showed that a combination of 3D-microgravity bioreactor using micro-carrier can be the best system for protein production using baculovirus in the mammalian cells.

目 錄
指導教授推薦書
口試委員會審定書
授權書…………………………………………………………………..III
誌謝……………………………………………………………………..IV
中文摘要………………………….……………………………………V
英文摘要………………………….……………………………….…VII
目錄…………………………………………………………………...VII
圖表目錄…………………………………………………………..…XIII
第一章 緒論…………………………………………………………..…..1
1.1前言……………………………………………………….……..1
1.2研究目的………………………………………………….……..2
第二章 文獻回顧…………………………………………………….…..3
2.1生產外源蛋白質各系統簡介………………….………………..3
2.1.1 微生物生產系統…….…………………………….……….3
2.1.2 植物生產系統…………………………….………………..4
2.1.3 動物生產系統…………..…………...……………………..5
2.1.4動物細胞生產系統…………………………………………6
2.1.5昆蟲系統……………...…………………………...…..……7
2.1.5.1桿狀病毒－昆蟲細胞生產系統………..….....………..7
2.1.5.2桿狀病毒－昆蟲幼蟲生產系統…..…………………...8
2.1.5.3桿狀病毒－蟲蛹生產系統…….…………………...…9
2.2桿狀病毒的架構...…………………………….………………...9
2.2.1桿狀病毒的分類……...…………………………………….10
2.2.2桿狀病毒生活史……………….…………………………...11
2.2.3 AcMNPV的基因表現……………………………………...13
2.2.4 桿狀病毒表現載體架構…………………………………..14
2.2.5桿狀病毒-昆蟲細胞生產系統之應用……………………...16
2.2.6桿狀病毒-哺乳類動物細胞生產系統……………………...17
2.2.7穿梭啟動子(Shuttle promoter)…………………………....18
2.3動物細胞及昆蟲細胞培養技術………………………………...21
2.3.1.1貼附式培養…………………………..…………………...21
2.3.1.2懸浮培養………………………………………..………...22
2.3.2培養製程………….………………………………………...22
2.3.2.1批次培養(Batch)……………........................................22
2.3.2.2流加培養(Fed batch)…………………………..……...23
2.3.2.3重複流加培養
(Repeatedfed batch orSemicontinuous)………………..……23
2.3.2.4連續培養－灌流式 (Continuouscultivation-perfusion)…………………..……...23

2.3.3動物細胞之固定化培養……….…………........….……...23
2.4固定化載體……………………………………….....................24
2.4.1絲瓜纖維……………….……………….…….…………...25
2.4.2微載體………………….………………………………...27
2.5生物反應器…………………………………………………...28
2.5.1攪拌式反應器………….…………….…………………...30
2.5.2 3D微重力反應器………………………………………...31
2.5.3膜反應器…………….……………………………….…...32
2.6實驗架構………………………………………………….....34
第三章 材料與方法……………………………………………………35
3.1 實驗儀器設備…..…………….……..……………………….35
3.2 實驗藥品………...….…....………………………………….36
3.3細胞株及病毒..……………………………………………….37
3.4 實驗方法..….…….…………………..……………………….38
3.4.1 病毒增殖………………………………………………….38
3.4.2 病毒力價測定……………………….……………………39
3.4.3 β-galactosidase活性檢測………..……………………..39
3.4.4 昆蟲細胞的懸浮培養….………………………………40
3.4.5桿狀病毒懸浮大量生產………………………………….41
3.4.6昆蟲細胞懸浮流加培養………………………………….41
3.4.7哺乳類動物細胞懸浮培養…….………………………….42
3.4.8細胞活性檢測……………………………………………..43
3.4.9哺乳類動物細胞固定化培養….………………………….43
3.4.9.1 Spinner flask…………...............…………………….43
3.4.9.23D-微重力反應器…………........…………………….44
3.4.10桿狀病毒轉染動物細胞…………...…………………….45
3.4.10.1 24 well........................……………………………….45
3.4.10.2 Spinner flask.............…………........……………….45
3.4.11病毒多次感染動物細胞…………………………………46
3.4.11.1 24 well......................………..........………………….46
3.4.11.2 Spinner flask...........……………..........…………….47
3.4.11.3 在Spinner flask中進行microcarrier的固定
化培養..................................................................………….47
3.4.11.4 在3D-微重力反應器中進行microcarrier的固
定化培養...............................................................................50
第四章 結果與討論…………………………………………………....52
4.1昆蟲細胞懸浮培養最適化探討…………………………..……53
4.2昆蟲細胞懸浮流加培養討 …………..…..…..………………...54
4.3最佳感染病毒力價(moi)探討………………..….……………..57
4.4病毒感染時機探討…………………………………………….61
4.5流加培養昆蟲細胞進行桿狀病毒之生產………………..…...61
4.6桿狀病毒感染哺乳動物細胞之轉染時間探討..…….…...…...62
4.7桿狀病毒感染哺乳類動物細胞之轉染病毒力價探討…….....64
4.8桿狀病毒感染哺乳類動物細胞之時機探討……………….....66
4.9桿狀病毒流加感染哺乳類動物細胞………….……………...67
4.10桿狀病毒流加感染哺乳類動物細胞次數最適化探討……...69
4.11桿狀病毒流加感染哺乳類動物細胞之反應器生產測試…...71
4.12哺乳類動物細胞固定化培養生產外源蛋白………………...73
第五章 未來展望………………..…………………………………......75
參考文獻………………………………………………………………..76


圖 表 目 錄

Figure 2.1不同病毒力價感染昆蟲細胞與β-gal活性關係圖…….....20
Figure 2.2不同病毒力價感染哺乳類動物細胞與β-gal活性關係圖.20
Figure 2.3絲瓜纖維外觀與剖面照……………………………..…...27
Figure 2.4以鐵絲懸掛固定LS與PU之spinner flask………………..29
Figure 2.5盤狀旋轉式生化反應器與滾筒狀旋轉式反應器之架構....31
Figure 2.5盤狀旋轉式生化反應器與滾筒狀旋轉式反應器之規格....31
Figure 2.7膜反應器……………………………………………..……..32
Figure 2.8填充床反應器………………………………………..……..33
Figure 4.1實驗架構…………………………………………….....…...53
Figure 4.2不同轉速與不同PF-68含量之昆蟲細胞生長曲線……...55
Figure 4.3昆蟲細胞流加懸浮培養細胞濃度探討………………..…..56
Figure 4.4昆蟲細胞流加懸浮培養細胞活性探討………………..…..56
Figure 4.5不同感染力價(moi)與病毒產量關係圖……………..........58
Figure 4.6病毒感染最佳時機………………………..….....................60
Figure 4.7流加培養昆蟲細胞生產桿狀病毒…………………..……..61
Figure 4.8病毒感染哺乳類動物細胞時間與細胞活性關係圖……....63
Figure 4.9病毒感染力價與β-gal產量關係圖…….……………........65
Figure 4.10病毒感染哺乳類動物細胞之時機探討……….....………66
Figure 4.11病毒感染方式與β-galactosidase產量關係圖…….……68
Figure 4.11桿狀病毒感染哺乳類動物細胞次數最適化探討…..……70
Figure 4.13桿狀病毒流加感染哺乳類動物細胞之反應器生產測試..72
Figure 4.14哺乳類動物細胞固定化培養生產外源蛋白……………..74

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