抗血管内皮生长因子受体2单链抗体的筛选及表达纯化

The Screening,Expression and Purification of Single Chain Fv(scFv) against Vascular Endothelial Growth Factor Receptor 2

为了竞争性抑制血管内皮生长因子与血管内皮生长因子受体2的结合作用,采用噬菌体展示技术,从噬菌体抗体库中筛选得到血管内皮生长因子受体2的高亲和力抗体,再通过PCR分析,基因测序,序列比对等步骤挑选出高亲和力的抗体片段的基因序列AK404,转化至大肠杆菌HB2151中,大量发酵进行可溶性表达,将表达产物通过镍柱纯化,最后经SDS-PAGE鉴定,获得纯度为95%的电泳纯抗体,产量可以达到每升发酵液614μg。定量ELISA分析显示,AK404与受体的结合呈剂量依赖性,进一步研究开发可以用于抗血管生成抗肿瘤的治疗或肿瘤诊断。

To block the activity of vascular endothelial growth factor transmitted via vascular endothelial growth factor receptor 2 pathway, a high affinity antibody against vascular endothelial growth＇factor receptor 2 was screened from phage display antibody library. After PCR analysis, DNA sequencing, and sequence identification, the gene of selected antibody was transformed into E. coli HB2151. Soluble ScFv antibody was expressed in the recombinant E. coli HB2151 induced by lmmol/L IPTG and further purified by Ni-IMAC. The yield was 614μg/L with purity of 95%. In conclusion, a scFv named AK404 was obtained which will potentially serve as drug candidate in anti-angiogenesis tumor therapy or tumor diagnosis.