HEK293F 细胞瞬时表达 PDGFR-β／Fc蛋白的条件优化

摘要

To optimize the conditions for transient transfection in HEK293F cells and improve the pro-duction of PDGFR-β,DNA (pXLG-PDGFR-β) and PEI were separately diluted in ultra-pure water, mixed together,and incubated at room temperature.The PEI-DNA complex was then added to the cells, and the culture was incubated at 37 ℃with 6% CO2 with agitation at 180 r/min,then incubated for 7 days.The PDGFR-β/Fc concentration in the culture medium was determined by sandwich ELISA.The conditions including cell density,DNA concentration and ratio of DNA to PEI,as well as other effectors such as adding of VPA and peptone were optimized.The results showed that transient gene expression yields of PDGFR-β/Fc can be maximized under following conditions:4 ×106 cells/mL,2.0 μg/106 cells DNA,1∶2 ratio of DNA and PEI and polymerize 5 minutes.The productivity can be further increased with adding of 3 mmol /L VPA,glucose and 1 g/L TN1.The experiment showed that when the conditions for transient transfection of HEK293F cells were optimized,the PDGFR-β/Fc yields up to 55 mg/L were achieved at the conditions,which laid a foundation of PDGFR-β/Fc expression for larger scales transfec-tion in HEK293F cells.%为优化 HEK293F 细胞瞬时转染条件，提高 PDGFR-β的蛋白表达量，采用聚乙烯亚胺（Polyetherimide， PEI）为转染试剂，将质粒 pXLG-PDGFR-β／Fc 与 PEI 混匀聚合后加入到细胞悬液，37℃，φ＝6％ CO2，180 r／min 悬浮振荡培养，培养7 d 后收集样品，ELISA 检测 PDGFR-β蛋白的浓度。对细胞密度、DNA 浓度和 m （DNA）：m （PEI）、聚合时间以及添加物（丙戊酸钠、葡萄糖和蛋白胨）进行优化。结果显示，HEK293F 细胞瞬时转染的最佳条件为：细胞密度4×106 cells／mL、DNA 浓度2.0μg／106 cells、m （DNA）：m （PEI）为1：2、聚合时间5 min。同时，48 h 内添加3 mmol／L 丙戊酸钠，第3天补加葡萄糖和1 g／L 的蛋白胨 TN1可使 PDGFR-β／Fc 的蛋白表达量明显提高。实验表明，经过对 HEK293F 细胞瞬时表达 PDGFR-β的转染条件的优化，蛋白表达量可达55 mg／L，为更大规模瞬时转染生产 PDGFR-β／Fc 蛋白奠定了基础。