Abstract
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.
Original language | English |
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Article number | 14 |
Number of pages | 10 |
Journal | Cell and Bioscience |
Volume | 4 |
Issue number | 1 |
DOIs | |
Publication status | Published - 2014 |
Open Access - Access Right Statement
© 2014 Liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Keywords
- eukaryotic cells
- genes