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Xinyuan Zhang; Characterization and function of a soluble VEGF receptor 2 protein. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2968.
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© ARVO (1962-2015); The Authors (2016-present)
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 and further to evaluate in vivo.
HEK293 cells were purchased from the Cancer Institute of the Chinese Academy of Medical Sciences Cell Bank. The E. coli DH5α strain was from our laboratory. The plasmid pCMV6-rVegfr2 was constructed according to the nucleotide sequence published in GenBank (GenBank accession number NM_013062.1). pCMV6-rVegfr2 was truncated with the restriction endonuclease EcoRVT. Transient transfection and expression were performed according to the standard protocol. ELISA was applied as previously described.
pCMV6-trunctated-rVegfr2 (6100 bp) was successfully cloned. The transfection experiments showed that eitherpCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein. 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. 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.
The use of transient transfection technology greatly improved transfect efficiency. Recombinant sVEGFR2 inhibited the effect of endogenous full-length VEGFR2 but was not cytotoxic.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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