Cloning And Expression Of Human Soluble Vascular Endothelial Growth Factor Receptor 1(sFlt-1) Gene In S. Lividans And P.pastoris And Purification Of SFlt-1

VEGF (Vascular Endothelial Growth Factor) is one of the growth factors that bind with heparin. It has specific mitogenic effects on vascular endothelial cells and can promote the proferliation of vascular endothelial cells and formation of new vascular. It also increases the permeability of vascular. All these biological effects are exerted with its specific receptors (VEGFRs). There are three kinds of VEGFRs: VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt-4.The gene of Flt-1 spans 7680 bp. Its encoding sequence of 4014 bp encodes 1338 amino acids. Flt-1 is composed of extramembrane, transmembrane and intramembrane regions. The extramembrane region consists of 758 aminoacids, including signal peptide containing 31 amino acids at N-terminus and seven Ig-like domains. After VEGF binds with the ligand-binding cluster locating in the second Ig-like domain, the tyrosine kinase of Flt-1 is activated, futhermore transducts the signals which regulate the migration and adhesion of endothelial cells. It also regualtes the signal of proferliation signal via KDR.Using RNA extracted from human umbilical vein endothelial cell as a template, the cDNA encoding the Ig-like domainl~4 of Flt-1 was amplified by RT-PCR. The cDNA was cloned into plasmid pUC18 and sequenced. The results showed that there were two A—G mutations at 850 bp and 1168 bp, which occurred in the domain 3 and domain 4, resepectively. Both the mutations didn't affect the biological activity of sFlt-1.Firstly, cloning and expression of sFlt-1 gene was performed in S.lividans TK24. Two different signal peptides vsi and gpp were used to investigate the secretory expression efficiency in S.lividans. The sFlt cDNA was inserted into the downstream of gpp signal peptide in the plasmid pSGLgpp. At meantime, the vsi sequence was fused with sFlt cDNA in the right ORF. The fusion genes were inserted into the shuttle plasmid pUWL-219. Recombinant plasmids were transformed into S.lividans TK24, respectively. The two recombinant strains named as S.lividans[pSGLgpp-F] and S.lividans[pUWL219-vsi-F] were obtained.