CAJ | 학술논문

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。
위우화 HEK293F 세포순시전염조건，제고 PDGFR-β적단백표체량，채용취을희아알（Polyetherimide， PEI）위전염시제，장질립 pXLG-PDGFR-β／Fc 여 PEI 혼균취합후가입도세포현액，37℃，φ＝6％ CO2，180 r／min 현부진탕배양，배양7 d 후수집양품，ELISA 검측 PDGFR-β단백적농도。대세포밀도、DNA 농도화 m （DNA）：m （PEI）、취합시간이급첨가물（병무산납、포도당화단백동）진행우화。결과현시，HEK293F 세포순시전염적최가조건위：세포밀도4×106 cells／mL、DNA 농도2.0μg／106 cells、m （DNA）：m （PEI）위1：2、취합시간5 min。동시，48 h 내첨가3 mmol／L 병무산납，제3천보가포도당화1 g／L 적단백동 TN1가사 PDGFR-β／Fc 적단백표체량명현제고。실험표명，경과대 HEK293F 세포순시표체 PDGFR-β적전염조건적우화，단백표체량가체55 mg／L，위경대규모순시전염생산 PDGFR-β／Fc 단백전정료기출。
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.