Transient gene expression of soluble VEGFR2: I-IV / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 810-816, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-342832
ABSTRACT
The extracelluar domain I-IV of target gene VEGFR2 (Vascular endothelial growth factor receptor 2) was cloned from villus of trimester abortion by RT-PCR, and linked to the expression vectors. Then, the transfection conditions were optimized in serum-free suspension culture HEK293 using GFP (Green fluorescence protein) as the report gene. The results showed that the optimal transfection efficiency and cell number were obtained when the ratio of foreign DNA PEI = 12 (W/W), DNA = 1.5 g /10(6) cells and shaking speed (120 r/min) in serum free medium in the beginning 4 hours of transfection. After optimizing the transfection conditions, the expression vector was successfully constructed for transient gene expression in HEK293, COS-7, and CHO-K1. The result shows that the target protein was only detected in CHO-K1 supernatant. Because of the C-terminal 8-His tag of target protein, target protein was subsequently purified using Ni2+-IDA and 5 mg purified protein was obtained in 1.5 L supernatant of CHOK1.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Solubility
/
Recombinant Proteins
/
RNA, Messenger
/
Transfection
/
Cell Line
/
Gene Expression Regulation
/
Chorionic Villi
/
Cricetulus
/
Culture Media, Serum-Free
/
CHO Cells
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article
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