暨南大学生命科学技术学院∥广东省生物工程药物重点实验室∥基因工程药物国家工程研究中心,广东,广州,510632
纸质出版日期:2015,
网络出版日期:2015-9-25,
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洪坡, 袁凤媚, 黄嘉慧, 等. HEK293F细胞瞬时表达PDGFR-β/Fc蛋白的条件优化[J]. 中山大学学报(自然科学版)(中英文), 2015,54(5):96-101.
HONG Po, YUAN Fengmei, HUANG Jiahui, et al. Condition Optimization of PDGFR-β/Fc Transient Expression in HEK293F Cells[J]. Acta Scientiarum Naturalium Universitatis SunYatseni, 2015,54(5):96-101.
为优化HEK293F细胞瞬时转染条件,提高PDGFRβ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% CO
2
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4×10
6
cells/mL、DNA浓度2.0 μg/10
6
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PDGFRβ/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PDGFRβ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PDGFRβ/Fc蛋白奠定了基础。
To optimize the conditions for transient transfection in HEK293F cells and improve the production of PDGFR-β
DNA(pXLG-PDGFR-β)and PEI were separately diluted in ultra-pure water
mixed together
and incubated at room temperature. The PEIDNA complex was then added to the cells
and the culture was incubated at 37 ℃with 6% CO
2
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:4×10
6
cells/mL
2.0 μg/10
6
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 transfection in HEK293F cells.
PDGFR-&beta/FcHEK293F细胞瞬时表达
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