血管内皮细胞生长因子受体Flt-1结合小肽的融合表达及活性鉴定

血管内皮细胞生长因子 (VEGF)通过结合其酪氨酸激酶受体KDR、fms样酪氨酸激酶 1(Flt 1)调节新生血管形成 ;筛选能封闭VEGF结合Flt 1的小肽 ,可以通过阻断肿瘤血管形成 ,抑制实体瘤生长 .将从噬菌体 12肽库中筛选获得的 2个能与Flt 1结合的阳性噬菌体克隆 (F5 6和F90 )十二肽DNA(36bp)克隆到表达载体pQE4 2中 ,在大肠杆菌M15中稳定表达二氢叶酸还原酶融合蛋白(DHFR F5 6 F90 ) ,经变性、复性后得到纯度达 90 %的可溶性蛋白 .ELISA检测表明 ,DHFR F5 6 F90能结合可溶性受体sFlt 1和血管内皮细胞 ;12 5I VEGF竞争抑制实验显示 ,DHFR F5 6能竞争抑制VEGF同可溶性受体sFlt 1结合 .结果提示 ,F5 6可能是VEGF受体Flt 1的有效拮抗剂 ,具有抗肿瘤新生血管形成的潜在应用前景

Fusion Expression of Peptides Binding to VEGFR-1 and Identification of Their Biological Activities

Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt 1) induces angiogenesis. In previous work, two positive clones binding to VEGF receptor Flt 1 were selected from phage display peptide library. To identify the special binding activity, two oligonucleotide fragments synthesized according to the positive phage 12 peptide sequences were cloned into DHFR fusion protein expression vector pQE42 for their expression in E.coli M15. Soluble DHFR F56/F90 were obtained after denaturation and refolding. ELISA demonstrated that DHFR F56 /F90 were able to bind to sFlt 1 and vascular endothelial cells. Moreover, DHFR F56 completely abolished VEGF binding to sFlt 1. Taken together, these data demonstrate that F56 may be an effective antagonist of VEGF receptor sFlt 1, and a potent inhibitor of tumor angiogenesis too.