Expression And Preliminary Function Evalution Of The Analogs Of VEGF165and VEGF165b

The VEGF family includes two kinds of protein VEGF×××and VEGF×××b, such asVEGF165and VEGF165b, which are glycoprotein consisting of165amino acids, structurallysimilar but functionally opposite. VEGF165can promote endothelial cell proliferation, migrationand angiogenesis, resulting in a wide application prospect in vascular diseases, whileVEGF165b could inhibit some of these functions mediated by VEGF165, leading to apotential value in the application of antivascular diseases. Although VEGF165andVEGF165b show good prospects in clinical trials, the half-life of VEGF165is short in vivoand VEGF165b has lower binding affinity of its receptor VEGFR2，which limites theirapplication.Based on these problems, we developed our studies:To prolong half-life of VEGF165in vivo, we adopted albumin (HSA) fusion method.Recombinant expression plasmid pPIC9K-HSA-VEGF165constructed successfully waslinearized by SaI l and was transformed into Pichia pastoris GS115by electroporation toobtain GS115/HSA-VEGF165by MD plates screening. The protein， HSA-VEGF165，expressed by Pichia pastoris GS115was purified by Blue and SP column. SDS-PAGE showedthat the molecular weight of purified fusion protein HSA-VEGF165was about89Kda andthe purity of it was more than95%. Western Blot showed protein had good immunogenicity.Cell activity assay showed that HSA-VEGF165could promote the proliferation of HUVECcells. When serum concentration was6.6%and10%, EC50of HSA-VEGF165was262.2ng/mL and25ng/mL respectively，which indicated that the activity of HSA-VEGF165wassimilar to VEGF165. Half-life of HSA-VEGF165measured in mice increased5timescompared with VEGF165.In order to improve binding affinity of VEGF165b and VEGFR2receptor, based on theanalysis data of binding sites between VEGF165and VEGFR2receptor，we chosed the pointmutation of E44R, E67R, EE7273RR. VEGF165b mutant gene obtained by overlapping PCRamplification was inserted into pPIC9K expression vector, resulting in pPIC9K-VEGF165bmut (mut means: E44R, E67R, EE7273RR). Recombinant expression plasmidpPIC9K-VEGF165bmut was linearized by SaIl and was transformed into Pichia pastorisGS115by electroporation, successfully obtaining GS115/VEGF165bmut. The protein,VEGF165bmut, was purified by Ni column. SDS-PAGE and Western Blot showed themolecular of VEGF165bmut was about20~23Kda. The purity of recombinant protein is morethan95%and has also good immunogenicity. The binding affinity between endothelial cellsand VEGFR2receptor was determined by ELISA and showed that VEGF165E67R wasactually improved high but others not. When the concentration of VEGF165bE67R was640ng/mL, the inhibition rate of endothelial cell proliferation mediated by VEGF165is100%.Moreover, VEGF165bE67R also inhibited45%migration of A549cells by adding to360ng/mL.According to these research, it solved the shortages of VEGF165and VEGF165b in acertain extent, short half-time and binding affinity between VEGF165b and VEGFR2 respectively. It will provide a theoretical basis for VEGF165and VEGF165b in the clinicaltrials.