Expression and characterization of a soluble VEGF receptor 2 protein

Cell & Bioscience

Ningli Wang⁵  Tao Jiang⁴  Jianqiu Mou²  Donna Lai³  Shisan Bao¹  Ching Song²  Xinyuan Zhang⁵  Wei Liu² 

[1] Discipline of Pathology, D06, Bosch Institute, the University of Sydney, Sydney, NSW 2006, Australia;University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, P. R. China;Bosch Institute, F13, the University of Sydney, Sydney, NSW 2006, Australia;State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology (IGDB), No.1 West Beichen Road, Chaoyang District, Beijing 100101, China;Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing 100730, PR China

关键词: Transient transfection;    Gene construction;    Truncated soluble protein;    Gene transfection;   

Others  :  791028
DOI  :  10.1186/2045-3701-4-14

 received in 2014-01-31, accepted in 2014-03-04,  发布年份 2014

【 摘 要 】

Objective

To clone and express a truncated, soluble vascular endothelial growth factor receptor 2 (sVEGFR2) possessing the combined-functional domains 1–3 and 5 in eukaryotic cells and to test the inhibitory effects of full length VEGFR2 in vivo.

Results

pCMV6-trunctated-rVegfr2 (6100 bp) was successfully cloned. The transfection experiments showed that either pCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein, which is usually used as an exogenous transfected reporter gene to determine the transfected efficiency. An analysis of the transfected cells revealed that the amount of full-length VEGFR2 protein in the pCMV6-truncated-rVegfr2 transfected cells was 20% lower than that in the negative control (non-transfected HEK 293 cells). The differences in test results between the transfected and negative control groups were greatest from 24–30 h after transfection; this period was therefore chosen as optimal for collecting culture supernatants. This analysis was highly sensitive for detecting the amount of sVEGFR2 protein expressed and secreted by the cells, and the sVEGFR2 protein content was found to increase by approximately 26% in the transfected cells compared to that in the negative control cells (68.2% ± 1.7% vs. 41.9% ± 2.9%, P = 0.000) and by 18% compared to the negative control cells (68.2% ± 1.7% vs. 50.0% ± 0.5%, P = 0.003). Propidium iodide and Hoechst staining indicated no significant change in the number of HEK293 cells undergoing apoptosis 6 days after pCMV6-trucated-Vegfr2 transfection, compared to the negative control. Soluble VEGFR2 produced by pCMV6-truncated-rVegfr2 inhibited full-length VEGFR2 protein expression in the cell membrane.

Conclusions

This study employed a eukaryotic system to express sVEGFR2. The use of transient transfection technology greatly improved transfect efficiency. Recombinant sVEGFR2 inhibited the effect of endogenous full-length VEGFR2 but was not cytotoxic.

【 授权许可】

   
2014 Liu et al.; licensee BioMed Central Ltd.

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