HEK293F细胞瞬时表达PDGFR-β/Fc蛋白的条件优化

Condition Optimization of PDGFR-β/Fc Transient Expression 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p XLG-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4×106cells/m L、DNA浓度2.0μg/106cells、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。

To optimize the conditions for transient transfection in HEK293 F cells and improve the production of PDGFR-β,DNA（ p XLG-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 7days. 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 / m L,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 HEK293 F 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 transfection in HEK293 F cells.