抗KDR单链抗体阻断VEGF/KDR通路的活性研究

Research on Inhibition Activity of a Single Chain Fv (scFv) against Vascular Endothelial Growth Factor Receptor 2

为了阻断VEGF与KDR的结合,抑制VEGF结合KDR后介导的内皮细胞迁移和血管新生,实验室已经从全人源噬菌体展示抗体库中筛选得到几种抗KDR的单链抗体,从中挑选一种亲和力较高的抗体命名为AK506进行周质表达,纯化后经SDS-PAGE鉴定,可获得纯度为95%的电泳纯抗体,产量可以达到每升发酵液1 mg。经表面等离子共振技术测定,AK506与KDR胞外区相互作用的平衡解离常数为7.99×10-9nmol/L。细胞ELISA分析显示,AK506与内皮细胞表面膜受体KDR的结合呈剂量依赖性。采用细胞划痕损伤修复实验和鸡胚尿囊膜实验证明,AK506可以显著抑制内源性VEGF诱导的血管内皮细胞迁移和新血管生成,具有进一步研究开发用于抗肿瘤血管生成治疗的潜力。

In order to block the binding of vascular endothelial growth factor（VEGF） with vascular endothelial growth factor receptor 2（KDR）,some single chain Fv（scFv） antibodied against KDR from full human phage display antibody library has been biopanned.One of them named AK506 which with higher affinity was expressed into the periplasmic space and purified by Ni-IMAC.The yield was 1 mg/L with purity of 95%.The surface plasmon resonance（SPR） technique and cell ELISA assay were adopted to test the interaction between AK506 and KDR,of which equilibrium dissociation rate constant（KD） was 7.99 nmol/L.The inhibition activities of AK506 on VEGF-induced endothelial cell proliferation and migration were studied using MTT assay and scratch-wound healing assay.Chorioallantoic membrane（CAM） assay was adopted to observe the antiangiogenic activity of AK506.The results showed that AK506 could bind with KDR on the surface of HUVEC cells and inhibit the binding of VEGF with KDR.According to these,AK506 can serve as a useful drug candidate in research,diagnostics and therapy of anti-angiogenesis tumor therapy.