小鼠天然可溶性VEGF受体基因sflt-1的克隆及鉴定

 可溶性血管内皮细胞生长因子受体 (sFlt 1 )与膜表面受体Flt 1竞争结合血管内皮细胞生长因子 (VEGF) ,并且与膜表面受体Flt 1及KDR形成异源二聚体 .完全阻断VEGF的生物学活性 ,除与sFlt 1的结合部位结构域有关外 ,还与整个蛋白质分子的高效分泌表达有关 .而蛋白质分子的高效分泌表达与蛋白质在细胞内高尔基体及内质网的加工密切相关 ,基因工程重组可溶性受体由于一些尚未明了的原因 ,往往不能高效表达 ,从而大大影响了其应用价值 .利用RT PCR技术从胚胎小鼠中扩增出天然可溶性VEGF受体基因sflt 1 ,克隆于pcDNA3载体中 ,在COS 7细胞中短暂表达 ;并克隆至pET42b载体中 ,经IPTG诱导后 ,可大量稳定表达与His Tag形成的融合蛋白 ,经HisNi柱纯化 ,可特异性结合VEGF .可溶性受体sFlt 1在肿瘤组织的高效表达可有效阻断新生血管的形成 ,从而为肿瘤的治疗探索一种方法 .

Cloning and Identification of Mouse Natural Soluble Vascular Endothelial Growth Factor Receptor

The increasing data show that the soluble form of Flt\|1 have the ability to block the biological activity of VEGF due to competitive binding of VEGF and formation of homologous and heterogenous dimmer with transmembrane Flt\|1 and KDR.However,for the complete blocking of the biological activity of VEGF,it depends on highly efficient expression of sFlt\|1 besides its high affinity to VEGF.The highly efficient expression of protein is closely related with the processing in Golgi body and endoplasmic reticulum.The recombinant soluble Flt\|1 has a relative lower expression than natural soluble Flt\|1 in eukaryote cell for unknown reason.So,how to increase the expression of soluble Flt\|1,becomes an urgently resolved problem.cDNA of natural soluble Flt\|1( sflt \|1) was cloned from the embryo of 12E days mouse by RT\|PCR.Then,the sflt\|1 gene was subcloned into pcDNA 3 eukaryotic expression vector.The Southern blot,Norhern blot and Western blot were performed in COS\|7 cells transfected with pcDNA 3\| sflt \|1 DNA.SDS\|PAGE showed that the protein of sflt \|1 could be well expressed.Moreover,the sflt \|1 gene was subcloned into prokaryotic expression vector pET42b,the sFlt\|1 fusion protein with His\|tag could be expressed abundantly after induction by IPTG.After denaturation,purification with His\|Ni column and refolding,the biological activity assay of sFlt\|1 was performed by inhibition on proliferation of HUVEC induced by VEGF.Because the high expression of sflt\|1 in tumor tissue could efficiently block the neovascularization,it would become a promising approach to tumor treatment.