血管内皮细胞生长因子受体(KDR)的分子克隆与原核表达

血管内皮细胞生长因子（ＶＥＧＦ）是特异的血管内皮细胞促分裂素，它主要通过相应受体（ＫＤＲ）刺激血管内皮细胞增殖．ＶＥＧＦ及其受体在肿瘤血管形成中起重要作用．通过逆转录及多聚酶链式反应（ＲＴ－ＰＣＲ）成功地从人脐静脉内皮细胞扩增出编码ＫＤＲ胞外ＶＥＧＦ结合区的ＤＮＡ片段，将该片段克隆在谷胱甘肽转移酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ２Ｔ中，获得在大肠杆菌Ｊｍ１０９的稳定表达．表达的不溶性融合蛋白可经碱变性法大量提取，为后续的研究工作奠定了基础．

Molecular Cloning of Vascular Endothelial Growth Factor Receptor(KDR)and its Prokaryotic Expression

Vascular endothelial growth factor(VEGF)is the highly specific mitogen for vascular endothelial cells,and it stimulates endothelial cell proliferation mainly through VEGF receptor(KDR).Both VEGF and KDR play an important role in tumor angiogenesis.To obtain highly expressed KDR in Vitro and prepare the antibody against KDR for blocking the funcation of VEGF,RT PCR was used to clone KDR cDNA fragment from human umbilical vein endothelia cells.The cloned cDNA was identified with sequencing and then cloned into fusion protein expression vector pGEX2T for its expression in Ecoli Jm109.After inducing by IPTG,the positive transformed bacteria with plasmid pGEX2T KDR can express stably GST KDR fusion protein.Molecular weight of KDR fusion protein is 60 kD,and the proportion of expressed KDR in total bactera protein is about 10%.The basic denatured protocol was used to extract the insoluble expressed proteins which will be used for preparing KDR antibodies.