To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Solubility ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-1 ; genetics ; isolation & purification ; metabolism