人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体研制与生物学活性测定

Development of monoclonal antibodies against extracellular domain Ⅲ of vascular endothelial cell growth factor receptor Ⅱ and its biological activity

根据GenBank中发表的血管内皮细胞生长因子(VEGF)受体Ⅱ(KDR)基因序列,设计1对引物经RT-PCR从人脐静脉内皮细胞中克隆出KDR胞外Ⅲ区(KDRD3)编码序列,克隆入原核表达载体pGEX-6P1。重组质粒转化大肠杆菌BL21,用IPTG于37℃条件下诱导,经SDS-PAGE和Western blotting分析,表达出的融合蛋白大小为33.4 ku。利用纯化的重组融合蛋白免疫BALB/c小鼠,经淋巴细胞杂交瘤技术筛选获得2株KDR特异性单克隆抗体C4和F6。2株单抗均能特异性识别HUVEC细胞表达的天然KDR分子,HUVEC迁移抑制试验和HUVEC体外血管形成抑制试验结果显示,2株单抗均能特异性阻断血管内皮细胞生长因子与KDR分子的相互作用。结果表明:所获得的2株单抗对KDR具有良好的特异性和中和活性。

A pair of primers was designed according to published gene sequence of human vascular endothelial cell growth factor receptor Ⅱ（KDR）.The coding sequence of KDR extracellular domain Ⅲ（KDRD3） was then amplified from human umbilical vein endothelial cells（HUVEC） by RT-PCR and cloned into prokaryotic expression vector pGEX-6P1.The recombinant plasmid was transformed into E.coli BL21 and induced with IPTG at 37 ℃.As evidenced by SDS-PAGE and western-blotting,recombinant GST-KDRD3 fusion protein was successfully expressed in E.coli.BALB/c mice were immunized with puried GST-KDRD3 fusion protein,and two KDR specific hybridoma strains were developed using hybridoma technique.Both mAbs could react with natural KDR expressed in HUVEC.The results of HUVEC migration inhibition test and in vitro angiogensis inhibition test showed that interaction between VEGF and KDR could be blocked by any one of KDR mAbs.Therefore,the obtained mAbs have good specificity and neutralization activity to KDR.