以水稻懸浮培養細胞蛋白質生產系統生產mGMCSF; Production of the mGMCSF in rice cell suspension culture

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题名:	以水稻懸浮培養細胞蛋白質生產系統生產mGMCSF;Production of the mGMCSF in rice cell suspension culture
作者:	賴穎慧;Ying-Hui Lai
贡献者:	生命科學研究所
关键词:	蛋白質高效率分泌系統;protein secretory system
日期:	2009-01-12
上传时间:	2009-09-22 10:21:05 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	自從1970年成功的利用大腸桿菌生產胰島素後，越來越多種醫藥蛋白質正開發中，除了微生物以外還有真菌、昆蟲、哺乳類動物…等生物都被作為表達系統。近年來，植物表達系統因為生產成本低，具有完整轉譯後修飾作用，且較無安全性顧慮等特性，已經被應用於生產許多不同的蛋白質，其中水稻細胞生產系統為重要的植物表達系統之一。為了讓我們的水稻表達系統生產更有效率，我們在目標蛋白質的N端附加上一段具分泌特性的信號肽，藉此將目標蛋白質運送至細胞外。我們選用了水稻蛋白質αAmy3、CIN1(cell wall invertase1)以及33kD的信號肽分別接上GFP作為報導基因以及mGMCSF作為我們的目標基因，並建立GFP以及mGMCSF分別融合三組信號肽的T1轉植水稻細胞系。利用西方點墨法偵測蛋白質在細胞中以及在液態培養基中的含量，並比較三種信號肽的分泌效率，分析的結果發現CIN1之信號肽在GFP和mGMCSF的轉植株中都有最佳的分泌效率，而33KD信號肽在轉植株中有次佳的分泌效率。另外，我們也得知Amy3啟動子中插入A1 intron片段比插入Ubi intron片段的具有更強表現mGMCSF 基因的能力。未來希望藉由集結這具有最佳分系效率的信號肽搭配最有效率的啟動子，以及最有效抑制懸浮培養系統中的蛋白水解酶的方式，發展一套高表現量的水稻細胞表達系統。 After the production of insulin by E. coli in 1970s, more and more therapeutical proteins were produced by different organisms, such as microorganism, insect and mammal. In the past few years, the plant expression system had been used to produce many kinds of recombinant proteins because of low cost, safety and post-translational modification. The rice cell production system is one of the most efficient plant expression systems. To improve the yield of the rice cell expression system, different signal peptides including αAmy3, CIN1 and 33kD have fused to the N-terminal of GFP or mGMCSF to explore the secretory efficiency of foreign proteins. Transgenic rice containing fusion proteins have been generated, and the intracellular and extracellular recombinant proteins were determined by western blotting to compare the secretory efficiency of three different signal peptides.. We found that CIN1 is the most efficient signal peptide for the secretion of both GFP and mGMCSF. On the other hand, we found that the addition of A1 intron in the 5’ end of fusion protein give rise to better secretory efficiency than the Ubi intron in the rice cell expression system. In the further, we would like to combine the most efficient signal peptide, the strong promoter and the inhibition of proteolysis to develop highly protein expression system in rice cells.
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