利用自裂解多肽T2A在牛耳皮肤成纤维细胞中实现多基因共同表达  

Co-Expression of Genes in Bovine Fibroblast Cells by Using Self-Cleaving Peptide T2A

作　　者：张俨[1] 马晴雯[1,2] 

机构地区：[1]上海交通大学医学院,上海市儿童医院,上海交通大学附属儿童医院,上海交通大学医学遗传研究所 [2]卫生部医学胚胎分子生物学重点实验室暨上海市胚胎与生殖工程重点实验室

出　　处：《生物技术通讯》2014年第3期361-364,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2011AA100602)

摘　　要：目的:探讨利用自裂解多肽2A构建的多顺反子载体能否在牛耳皮肤成纤维细胞中实现多基因的有效表达。方法:利用来自一点褐翅蛾病毒(TaV)的2A元件(T2A)将GFP和Neo基因连接到同一载体中,构建pCMV-GFP-T2A-Neo质粒,将其转染牛耳皮肤成纤维细胞,以FACS检测GFP基因的表达,RT-qPCR检测GFP、T2A和Neo的表达。结果:由T2A连接的GFP和Neo基因在mRNA水平上都有显著表达,且表达水平相当。结论:以T2A连接的基因在转入细胞后能正常翻译和表达,显示T2A在牛耳皮肤成纤维细胞中具有自裂解功能,可作为一种构建多顺反子载体的有效工具用于牛耳皮肤成纤维细胞的基因转移,为其将来在转基因牛研制中的应用奠定了基础。Objective： To investigate whether the muhicistronic cassettes linked by self-cleaving peptide 2A could be expressed efficiently in bovine fibroblast cells. Methods ＆ Results： A co-expression vector pCMV-GFP-T2A-Neo contains T2A from insect viruses TaV linked GFP and neomycin resistant genes was constructed and transfected bovine fibroblast cells. FACS analysis confirmed the expression of GFP. RT-qPCR results indicated that GFP, T2A and Neo expressed in bovine fibroblast cells with equal amount. Conclusion： T2A has self-cleav- ing activity in the bovine fibroblast cells which resulted in the expression of GFP effectively. It showed that T2A could be a potential tool for co-expression of proteins in the bovine fibroblast cells and may provide new clue for production of transgenic bovine with expression of multiple foreign genes.

关 键 词：多顺反子载体 自裂解多肽T2A 牛耳皮肤成纤维细胞 

分 类 号：Q78[生物学—分子生物学]