探討αAmy3、OsCIN1與Os33KD信號?在水稻懸浮培養細胞中的功能及特性; Characterized analysis of αAmy3, OsCIN1 and Os33KD signal peptide in rice cell suspension culture

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Title:	探討αAmy3、OsCIN1與Os33KD信號?在水稻懸浮培養細胞中的功能及特性;Characterized analysis of αAmy3, OsCIN1 and Os33KD signal peptide in rice cell suspension culture
Authors:	葉如芳;Ju-fang Yeh
Contributors:	生命科學研究所
Keywords:	信號肽;水稻懸浮培養細胞;signal peptide;rice cell suspension culture
Date:	2011-09-14
Issue Date:	2012-01-05 14:28:44 (UTC+8)
Abstract:	生物工程技術的應用有效地提供了重組蛋白質的生產。由不同類型的寄主细胞所生產的重組蛋白質可能會有不同的生物活性和蛋白質的穩定度，尤其是醫藥蛋白等高價值蛋白質。若要生產具有完整生物活性的高價值重組蛋白質，則以具備完整蛋白質後修飾作用的哺乳類動物細胞和植物寄主细胞作為生產平台之優先考量。在過去幾年，植物分子農場被認作為是一個兼具便利和濟策略表達外源蛋白質生產平台，例如抗體，醫藥蛋白，細胞素等等。水稻細胞生產系統為一有效率的重組蛋白質生產系统。為了要改良重組蛋白質在水稻細胞生產系統中之分泌效率，三種不同的信號肽包括αAmy3，OsCIN1 和Os33KD 融合到GFP 和mGMCSF 基因之N 終端序列，藉由分析轉殖細胞內外的重組蛋白質累積量，以探討三種信號肽之分泌效率。包含有三種不同信號肽的GFP 轉殖細胞株被建立，並且細胞內蛋白質和細胞外蛋白質由西方點墨法或FLUOROSK ANASCENTFL 來偵測重組蛋白質的累積量，而每一種信號肽之分泌效率在各自獨立的穩定轉殖水稻懸浮細胞株中進行比較。實驗結果顯示，Os33KD 信號肽相對其他兩者而言在GFP 水稻細胞懸浮培養有糖中有最高的分泌效率，而αAmy3 和OsCIN1 信號肽在缺醣處理下顯示了在缺醣情況下其分泌效率有被提升的現象。因此我們發現，在有糖誘導表現量較強的Ubiquitin 啟動子，使用αAmy3 和OsCIN1 信號肽會有較佳的分泌比例的表現。藉由對信號肽更多的探討及研究，我們能夠知道不同的生長環境可能會影響信號肽之分泌功能，深入認識信號肽之特性能夠幫助我們在不同的生長條件下選擇不同的信號肽，以期在植物細胞生產平台能夠達到更大量的重組蛋白質之產量。 Application of biotechnical approach has provided to be useful in the production of recombinant proteins. Recombinant proteins produced from different types of host cell have various biological activities and protein stability. To produce bioactive recombinant protein, mammal and plant host cells were used due to they have post-translational modification. In the past few years, plant molecular farming is considered as a convenient and economical production strategy to express foreign proteins, such as antibody, pharmaceutical medicine, cytokine, and so on. The rice cell production system is a useful recombinant protein production system. To improve the secretion efficiency of recombinant proteins in rice expression system, different signal peptides including αAmy3, OsCIN1 and Os33KD were fused to the N-terminal of GFP and mGMCSF respectively to explore the secretary efficiency of foreign proteins. Three different signal peptides fused to N-terminal GFP in transgenic rice cell lines were generated, and the intracellular proteins and extracellular proteins was detected by western blotting or FLUOROSKAN ASCENTFL. Therefore, the secretion efficiency of each signal peptide can be compared among different stable transformed rice suspension cell lines. The result showed that the Os33KD signal peptide has the highest secretion ratio in GFP transgenic rice cell suspension culture containing sucrose, but the αAmy3 and OsCIN1 signal peptides showed an increase in secretion during sugar starvation treatment. The efficiencies of signal peptides that are able to secret more recombinant proteins into culture medium is different according to the growth condition, therefore we can choose the suitable signal peptide in rice suspension cell bioreactor.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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