Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological...

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF(165). Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1% methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1 (2-3) to hVEGF(165) and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF(165) were close to those of sFlt-1(1-3). Animal test showed that sFlt-1(2-3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF(165) significantly.