臺灣博碩士論文加值系統

由於植物細胞具備多種生產重組蛋白質實用上的優點，因此，利用植物細胞來生產各種外源蛋白質，已逐漸受到廣泛的應用，尤其是工業或醫藥用高價值的蛋白質、疫苗或抗體之大量生產技術，均受到各國生技研發單位的重視並積極投入開發中。
在本研究中，欲開發出一套以水稻懸浮細胞為主體的生產技術平台，可快速且高效率生產我們所需的外源蛋白質。為了開發此一生產技術平台，口蹄疫次單位疫苗EVP1蛋白質被選為目標產物，以此系統來大量生產。本研究研發的重點包括：（1）水稻基因表現匣與載體的設計，可廣泛應用於各種不同類型的外源蛋白質之生產；（2）水稻細胞蛋白質胞外分泌模式的引入，可精簡產物純化的過程和花費；（3）開發新式酵素活性的產量指標分析系統，使量產程序的研發過程與監控分析簡易化；（4）建立快速、簡易的短暫分析法可篩選外源蛋白質表現量高之品系;（5）將轉殖水稻懸浮細胞進行再生，可得到後代種子以長久儲存優良品系。
本研究結果顯示，相對於須消耗冗長時間（6∼8個月）的傳統水稻轉殖系統，本系統可於短期（3∼4個月）的生產操作時間即可獲得EVP1，本研究初步結果EVP1之最高產量約為700 μg/L-medium，且高產量細胞品系可成功經由再生作用成為轉殖株。整合各項技術支援的連貫性，將使整體技術平台的研發成果具備相當的優勢和競爭力。

The research and development of scale-up production technology
of high-value proteins、vaccine or antibody is the most important issue in the whole biotechnology fields.
Plants and plant cells are now considered as viable and competitive expression systems for large-scale protein production. Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing, but the disadvantages are a farmland is needed. Problems of gene flow in field, and generation of transgenic plants is time-consuming. By contrast, plant-suspension cell culture can be used to produce recombinant proteins quickly, cost-effectively, avoid problems associated with biological safety of GM crop grown in filed, and easy scale-up by fermentation. In this study, we demonstrated rice suspension cells as a facile and economic bioreactor for the large-scale production of industrial and pharmaceutical recombinant proteins. We used the EVP1 vaccinogen protein of foot and mouth virus disease (FMVD) as a model. The expression construct was designed to encode a heterologous protein fused with an enzymatic reporter which could simplify the monitor in a process of protein yield. In the expression cassette, we chose the continuous expression GluB1 promoter to drive EVP1-APU target fusion protein expression; fuse a signal peptide gene for secretary system. Rice cells were transformed by Agrobacterium-mediated system. Selection and culture of transformed suspension cells could be generated within short time. We have developed a novel micro-well plate suspension cell cultured method to quickly select high protein expression cell lines, scale-up culture, and collect target proteins. In addition, the high protein expression cell lines could be regenerated into whole plants. The system we have developed could be operated with a high throughput method in a cost-effective and timely manner.

目錄
圖表目錄.................................................................................................... Ⅲ
縮寫字對照表........................................................................................... Ⅳ
中文摘要.....................................................................................................Ⅴ
Abstract..................................................................................................... VI
第一章 序論............................................................................................ 1
第二章 文獻回顧................................................................................... 2
2.1. 基因工程蛋白質製藥........................................................................... 2
2.2. 蛋白質表現系統................................................................................... 3
2.3. 植物細胞表現系統及水稻懸浮細胞轉殖系統................................... 9
第三章 研究目的、方向及方法........................................................ 12
3.1. 研究目的.............................................................................................. 14
3.2. 水稻懸浮細胞系統生產口蹄疫疫苗.................................................. 14
3.3. 研究方向及方法.................................................................................. 20
第四章 實驗材料與方法.................................................................... 28
第一節 生產EVP1-APU外源蛋白基因表現匣的構築及轉殖
4.1. 表現匣的設計與構築.......................................................................... 28
4.2. 接合反應.............................................................................................. 29
4.3. 細菌的轉殖作用.................................................................................. 31
4.4. 細菌plasmid DNA的純化.................................................................. 33
第二節 水稻懸浮細胞之農桿菌轉殖系統與培養系統的設立
4.5. 水稻細胞利用農桿菌（Agrobacterium）轉殖的方法...................... 34
第三節 高表現量單一轉殖水稻懸浮細胞品系（single cell line）
篩選與再生系統的設立
4.6. 轉殖水稻細胞之篩選及挑選高表現量之品系.................................. 38
第四節 外源蛋白分析方法
4.7. 細胞內蛋白質萃取方法...................................................................... 39
4.8. 培養液中蛋白質萃取方法.................................................................. 39
4.9. APU活性分析....................................................................................... 40
4.10.蛋白質濃度測定( Bradford法) ........................................................... 41
4.11.SDS-PAGE膠體的製備...................................................................... 41
4.12.蛋白質電泳(SDS polyacrylamide gel electrophoresis) ...................... 42
4.13.EVP1一級抗體製備........................................................................... 44
4.14.水稻懸浮細胞高濃度培養方法.......................................................... 45
4.15.西方墨點分析...................................................................................... 45
第五章 結果與討論............................................................................. 47
5.1. EVP1-APU融合重組蛋白質基因序列與基因表現匣轉殖載體的
建構...................................................................................................... 47
5.2. 水稻懸浮細胞之農桿菌轉殖系統的設立.......................................... 47
5.3. 獲得成功轉殖的水稻細胞族群.......................................................... 48
5.4. 從培養液中大量純化目標重組蛋白質的方法設立.......................... 48
5.5. 新式量產指標分析系統的設立.......................................................... 48
5.6. 發展快速、簡易的短暫分析法分離單一轉殖水稻懸浮細胞品系.. 49
5.7. 利用APU活性分析篩選表現量高之單一轉殖水稻細胞品系……. 50
5.8. 由高表現量水稻轉殖細胞品系培養液中分析目標重組蛋白質及
分析其產量.......................................................................................... 50
5.9. 高產量水稻轉殖細胞品系進行再生作用........................................ 53
第六章 結論…………........................................................................... 54
參考文獻.................................................................................................... 73
附錄............................................................................................................. 81
Ⅰ、EVP1 基因及蛋白質序列.................................................................... 81
Ⅱ、水稻轉殖流程........................................................................................ 82
Ⅲ、水稻細胞及組織培養之培養基成份配方............................................ 83
Ⅳ、碩士研究壁報發表研討會記錄............................................................ 88
Ⅴ、碩士研究口頭發表研討會記錄..................................................... 88
Ⅵ、碩士壁報發表英文摘要、口頭發表形式論文及其榮譽記錄........... 88

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