HEK293F细胞瞬时表达eGFP蛋白条件的优化
Optimization of transient gene expression of GFP in mammalian cell HEK293F in suspension
HEK293F细胞是最常用于表达重组蛋白药物的宿主细胞。哺乳动物细胞表达重组药物蛋白主要有两种方式：稳定基因表达（Stable Gene Expression）和瞬时基因表达（Transient Gene Expression）。与稳定基因表达相比，瞬时基因表达能在短时间内获得一定量的重组药物蛋白，以供早期临床研究使用。本实验采用瞬时基因表达方法，通过瞬时转染方法、细胞接种密度、质粒DNA浓度、DNA/PEI比例等条件进行优化，得出HEK293F细胞瞬时基因表达的最佳条件。为外源药用蛋白在HEK293F细胞中大规模瞬时表达打下基础。经过优化，得出HEK293F细胞瞬时表达GFP蛋白的最佳条件是：一步法的瞬时转染方法，4.0×106cells/mL细胞接种密度，2.0μg/1×106cells质粒DNA浓度，1:2 DNA/PEI比例。在此条件下细胞瞬时转染效率达到80%。
HEK293F cell is the most commonly host cell for expression of recombinant protein. There are two methods for production of recombinant protein in mammalian cells: Stable Gene Expression (SGE) and Transient Gene Expression (TGE). Compare whit SGE，TGE can obtain a certain amount of recombinant protein in a short time, for early stage clinical research . In this study，the factors influencing transfection efficiency, including transfected methods, amount of DNA, ratio of DNA and PEI, time of DNA/PEI complex formation, and cell density were optimized to express GFP in mammalian cell HEK293F in suspension. The optimum conditions is to lay the foundation for large-scale transient expression of recombinant protein in HEK293F cells. Optimized conditions, one-step transfected methods, 4.0×106cells/mL cell density , 2.0μg/1×106cells DNA concentration , 1:2 DNA/PEI complex Ratio were obtained. Under the optimized conditions，the transfection efficiency of HEK293f was up to 80%.
卢加、苏洪振、谢秋玲、沈虹宇、袁凤媚
细胞生物学分子生物学生物工程学
HEK293F细胞瞬时转染GFP荧光蛋白质粒DNAPEI一步法重组蛋白
HEK293F celltransient transfectionGFP fluorescent proteinDNA plasmidPEIone-steprecombination protein
卢加,苏洪振,谢秋玲,沈虹宇,袁凤媚.HEK293F细胞瞬时表达eGFP蛋白条件的优化[EB/OL].(2014-05-16)[2025-08-06].http://www.paper.edu.cn/releasepaper/content/201405-280.点此复制
评论