【摘要】 血管内皮生长因子受体(VEGFR)是血管内皮生长因子的特异性受体,VEGFR-2在介导VEGF刺激内皮细胞增殖及血管通透性等生物学活性中起重要作用。在大肠杆菌中实现可溶性的人血管内皮生长因子受体KDR胞外3区的表达,并鉴定其与配体结合的活性。采用重叠PCR的方法合成人血管内皮生长因子受体KDR胞外3区基因,将该基因与高效表达载体pET-32a重组,转化大肠杆菌Rosetta(DE3)中,表达产物依次经过CM阳离子交换树脂和镍柱亲和层析纯化。利用ELISA法和体外抑制VEGF刺激的人脐静脉内皮细胞增殖实验检测表达产物与配体结合的活性。SDS-PAGE显示,目的蛋白主要以可溶性Trx-KDR3融合蛋白表达于胞质,30oC时1 mmol/L IPTG诱导细菌5 h融合蛋白表达量约占胞质可溶性总蛋白的20%,经CM阳离子交换树脂和镍柱亲合层析纯化得到纯度为95%的产物,Western blotting鉴定是目的蛋白。ELISA和体外HUVEC细胞增殖实验显示,表达产物具有特异性结合hVEGF165的活性,且该作用呈一定的浓度依赖性。具有配体特异性结合活性的可溶性人血管内皮生长因子受体KDR胞外3区成功表达,为靶向血管抗肿瘤治疗和相关抗体的研究奠定了基础。更多还原

【Abstract】 Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessel.We prepared recombinant extracellular domain 3(KDR3) of human vascular endothelial growth factor receptor-2 in Escherichia coli and studied its specific binding activity with its ligand.The target DNA was synthesized by overlapping PCR,ligated with expression vector p-ET32a and transformed into E.coli Rosetta(DE3).The soluble fusion protein Trx-KDR3 was expressed in cytoplasm,which was up to 20% of total soluble protein in cytoplasm after having been induced by 1 mmol/L IPTG for 5 h at 30oC.It was characterized to be target protein by Western blotting.The product was purified by CM cation exchange resin and immobilized metal affinity chromatography(IMAC).Its VEGF-binding activity was determined by ELISA assay and its influence on the propagation of HUVEC induced by VEGF.The protein product showed high ligand binding activity in the ELISA and HUVEC propagation study compared to the control.Therefore,ligand binding active,soluble recombinant extracellular domain 3 of VEGFR-2 KDR was successfully expressed and purified in E.coli,which would be applied to anti-angiogenesis anti-tumor therapy and anti-KDR antibody development.更多还原