通过 CRISPR/Cas9 内含子靶向插入策略在水稻悬浮培养物中生产重组蛋白;Production of recombinant proteins in rice suspension cultures via a CRISPR/Cas9 intron-targeted insertion strategy

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Title:	通过 CRISPR/Cas9 内含子靶向插入策略在水稻悬浮培养物中生产重组蛋白;Production of recombinant proteins in rice suspension cultures via a CRISPR/Cas9 intron-targeted insertion strategy
Authors:	阮氏美;MAI, NGUYEN THI
Contributors:	生命科學系
Keywords:	CRISPR/Cas 9 敲入;水稻悬浮细胞;人 BMP2 重组蛋白;αAmy3 启动子;糖;CRISPR/Cas 9 knock-in;rice suspension cells;sugar;αAmy3 promoter;human BMP2 recombinant protein
Date:	2024-03-01
Issue Date:	2024-10-09 15:25:47 (UTC+8)
Publisher:	國立中央大學
Abstract:	農業生物技術是一系列工具，其中植物是理想的載體來表達藥物重組蛋白和食品。這項研究利用內源性αAmy3啟動子和信號肽來探討水稻懸浮培養細胞中生產生物活性製藥蛋白的效力。該αAmy3啟動子在無糖條件下受到強烈誘導，本研究用於生產人類骨形成蛋白2（mature hBMP2 , hBMP2m）。使用修改過的CRISPR/Cas9敲入（knock-in）系統，在重組基因的5’剪接位點，使得基因能夠插入到水稻αAmy3的內含子1中。在這項研究中，成功將重組的GFP基因或水稻hBMP2m序插入αAmy3的內含子1中，水稻轉職效率達12.5%及13.5%。在無糖培養基中處理的轉殖水稻懸浮細胞中，由於水稻αAmy3啟動子對缺糖反應的高活性和敏感性，重組基因的mRNA和重組蛋白的表達比利用農桿菌隨機嵌入染色體表達更高。原生αAmy3信號肽不僅將重組GFP蛋白雙重定位到澱粉體中，還引導GFP重組蛋白分泌到CRISPR介導的敲入水稻細胞系的培養基中。值得注意的是，rhBMP2m重組蛋白在每毫升細胞中產生了21.5μg，佔總可溶蛋白的1.03%，表示在αAmy3的rhBMP2m基因插入在兩代中保持一致。再者，來自水稻細胞的rhBMP2m蛋白表現出糖基化、二聚體形成能力和生物活性。我們的結果證明了以內源性水稻αAmy3啟動子-信號肽為基礎的表達系統對於生產重組蛋白質是有效的。這種修改後的CRISPR/Cas9基因敲入系統可以很容易地應用於水稻懸浮細胞培養中生產製藥蛋白質。;Agricultural biotechnology is a range of tools and plants are attractive platforms to express pharmaceutical recombinant proteins and food production. This study explores the efficacy of utilizing an endogenous αAmy3 promoter, which is induced strongly un-der sugar-free conditions, and signal peptide for the production of a bioactive pharma-ceutical protein, specifically the mature form of human bone morphogenetic protein 2 (hBMP2m) in rice suspension culture cells. The modified CRISPR/Cas9 knock-in sys-tem, which incorporates an artificial 3’ splicing site at the 5′ end of the recombinant gene, enables gene insertion into intron 1 of rice αAmy3. In this study, knock-in transgenic rice cell lines harboring the recombinant GFP gene or the rice-codon optimized mature form of hBMP2 cDNA (rhBMP2m) inserted in intron 1 of αAmy3 were generated with trans-formation efficiencies of 12.5% or 13.5%, respectively. Expression levels of recombi-nant genes were strongly upregulated by the high activity and sensitivity of the rice αAmy3 promoter in the transgenic rice suspension cells cultivated in a sugar-free liquid medium. Native αAmy3 signal peptide not only directed dual localization of recombi-nant GFP protein in amyloplasts but also guided the secretion of the GFP recombinant proteins into the culture medium in the CRISPR-mediated knock-in rice cell lines. Re-markably, the rhBMP2m recombinant protein yielded 21.5 μg/mL of cells, accounting for 1.03% of the total soluble protein, and showed consistent expression across two gen-erations of rhBMP2m gene knock-in at αAmy3 intron I. Furthermore, the rhBMP2m pro-teins derived from rice cells exhibited glycosylation, dimer formation and bioactivity. Our results demonstrate the effectiveness of the endogenous rice αAmy3 promoter–signal peptide-based expression system for producing recombinant proteins. This modified CRISPR/Cas9 knock-in system can be readily applied to the production of pharmaceuti-cal proteins in rice suspension cell cultures.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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