关键词： Antiangiogenic activity BSA Bovine serum album CAM Chorioallantoic membrane Dissociation E. Coli EC EGF ELISA Endothelial cells Enzyme linked immunosorbent assay Epidermal growth factor Equilibrium constant Escherichia coli FBS FGF Fetal bovine serum Fibroblast growth factors IFN-γ Interferon-γ K(a) K(d) KD KDR Kinase-insert domain-containing receptor (as it is called in human) Monoclonal antibody Periplasmic expression SPR Surface plasmon resonance TNF-α The kinetic constant for association Tumor necrosis factor-α VEGF VEGF receptor-2 VEGFR-2 VEGFR2 Vascular endothelial growth factor mAB

Mesh ： Angiogenesis Inhibitors / chemistry pharmacokinetics pharmacology Cell Movement / drug effects Cell Proliferation / drug effects Cells, Cultured Chorioallantoic Membrane / drug effects Endothelium, Vascular / drug effects Humans Peptide Fragments / chemistry pharmacokinetics pharmacology Protein Structure, Tertiary Solubility Transfection Vascular Endothelial Growth Factor A / antagonists & inhibitors pharmacology Vascular Endothelial Growth Factor Receptor-2 / chemistry

来  源：   DOI：10.1016/j.biopha.2013.06.001   PDF(Sci-hub)

Abstract：
Vascular endothelial growth factor (VEGF) is a key regulator of pathological angiogenesis and vascular permeability and overexpressed by most solid tumors. VEGF receptor-2 (VEGFR-2 or kinase-insert domain-containing receptor as it is called in human, KDR) is a specific receptor of VEGF with a high binding affinity. A solube recombinant extracellular domain 1-3 of human VEGFR-2 (rKDR1-3) was expressed in Escherichia coli (E. Coli) and purified from the bacterial periplasmic extracts by immobilized metal affinity chromatography and anion exchange chromatography to inhibit the VEGF-induced angiogenesis. A surface plasmon resonance (SPR) technology was adopted to analyze the affinity and kinetics constant between rKDR1-3 and VEGF165. Under the given experimental conditions, the association rate constant Ka was 1.06×10(5)M(-1) S(-1), the dissociation rate Kd was 6.09×10(-3) S(-1), the dissociation constant KD was 5.74×10(-8)M. The effect of rKDR1-3 on VEGF-induced endothelial cell proliferation was studied using MTT assay, scratch-wound healing assay and chorioallantoic membrane (CAM) assay. The results showed that rKDR1-3 could inhibit neovascularization and serve as a useful drug candidate in research, diagnostics and therapy of cancer.
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