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    請使用永久網址來引用或連結此文件: http://ir.lib.ncu.edu.tw/handle/987654321/6436


    題名: 利用水稻細胞之懸浮培養建立蛋白質高效率分泌系統;Development of a high efficient protein secretory system in rice cell suspension cultures
    作者: 劉馨如;Hsin-Ju Liu
    貢獻者: 生命科學研究所
    關鍵詞: 蛋白質高效率分泌系統;protein secretory system
    日期: 2006-09-12
    上傳時間: 2009-09-22 10:19:29 (UTC+8)
    出版者: 國立中央大學圖書館
    摘要: 近數十年來,有許多研究致力於利用不同的表達系統來生產具有醫療用價值的基因重組蛋白質。而一個理想的表達系統必須能以最低的成本來生產安全、具有生物活性的蛋白質。近年來植物的表達系統已然成為具有許多優勢的選擇,其中水稻系統的成本低,且不具毒性,目前已經應用於許多蛋白質的表現;除了選擇一個理想的表達系統之外,我們必須集結系統中的其他優勢使其更有效率。無論是真核或原核生物,蛋白質的信號肽已被許多研究利用來有效生產蛋白質藥物,使蛋白質的N端附加一段分泌信號肽,讓蛋白質得以分泌至細胞外,而簡化下游純化的工程並降低生產成本。動植物中的蛋白質信號肽有某些保守的特徵,但其氨基酸組成的變異大,因此不同蛋白質的信號肽被認為有不同的辨認機制,而使分泌效率有所不同。為了增加蛋白質在水稻細胞培養系統中的分泌效率,我們篩選水稻中的內生性的蛋白質信號肽,分別是αAmylase3、CIN1(cell wall invertase1)以及33kD,C端各別接上GFP (green fluorescent protein)作為報導基因,於水稻細胞中大量表現。利用西方點墨法偵測蛋白質並比較三種信號肽的分泌效率,分析的結果發現αAmylase3之信號肽有最佳的分泌效率。為了確定αAmylase3信號肽的分泌效率是否專一於GFP,抑或可以適用在其他蛋白質,因此我們將會分析這三組信號肽對於mGM-CSF (mouse granulocyte-macrophage stimulation factor)蛋白質的分泌效率。除了初步篩選水稻中的內生性信號肽,未來我們會從其他動植物,甚至酵母菌中進行篩選,希望集結這些資訊而使發展一套高分泌效率的水稻細胞表達系統。 Research in the past few decades has worked on the use of therapeutically valuable proteins from different protein expression systems. The ideal foreign protein expression system would be the one that produces the most safe, biologically active material at the lowest cost. Plant-based expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins. Most recombinant genes can be expressed in cultured rice cells; therefore, it is essential to determine which expression cassettes offer the most advantages for the production of the recombinant protein. The knowledge of protein signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. By adding a specific tag to the desired proteins, one can, for instance, tag them for excretion, making them much easier to harvest. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. To increase the secretion of recombinant protein in rice suspension cells, we generated overexpression vectors using different endogenous N-terminal signal peptides (αAmylase3,CIN1,33kD) fused with green florescence protein (GFP) and mouse granulocyte-macrophage stimulation factor (mGM-CSF), respectively. We detected the secreted GFP by Western blotting. Comparison of different signal peptides for secretion of GFP in rice suspension culture, we found that αAmylase3 is the better signal peptide for secreted the GFP out. To examine whether the supremacy of the αAmylase3 signal is specific for GFP, we’ll subsequently analyze the secretion efficiency of mGM-CSF and intend to develop a high efficiency secretion system in rice suspension cells.
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