重组人VEGF可溶性受体的表达纯化及结合性质初探

目的:表达优化的血管内皮细胞生长因子(VEGF)受体1(VEGFR1)胞外区第2个类免疫球蛋白结构域(VEGFR1D2)和VEGF受体2(VEGFR2)胞外区第3个类免疫球蛋白结构域(VEGFR2D3)与人IgG1 Fc片段的融合产物VEGF-Trap2,探讨该产物与人源VEGF165(hVEGF165)之间的亲和力。方法:将优化的目的基因VEGFR1D2/R2D3连接到真核表达载体pIRES2-EGFP-Fc中,转染CHO-K1细胞并筛选高表达目的蛋白VEGF-Trap2的细胞系,亲和纯化VEGF-Trap2蛋白,通过非竞争性ELISA及生物膜干涉技术检测VEGF-Trap2与hVEGF165之间的亲和力。结果:DNA测序表明真核表达载体pIRES2-EGFP-VEGF-Trap2序列正确;获得表达VEGF-Trap2的细胞系;非竞争性ELISA实验中,VEGF-Trap2与hVEGF165功能性亲和常数达到1.86×107L/mol;生物膜干涉实验中,hVEGF165与VEGF-Trap2的平衡解离常数达到3.13×10-9mol/L。结论:构建了真核表达载体pIRES2-EGFP-VEGF-Trap2并在CHO-K1细胞中稳定表达,重组蛋白VEGF-Trap2与hVEGF165有较高的亲和力,提示其可用于阻断VEGF信号传导途径,为该蛋白进一步的体外及体内实验奠定了基础。

Expression and Purification of Recombinant Human Soluble VEGF Receptor and Preliminary Study of its Binding Affinity

Objective： To investigate affinity between human VEGF165（hVEGFt65） and the expressed product of optimized the second immunoglobulin（Ig） domain of VEGFRI（VEGFR1D2） and the third Ig domain of VEGFR2 （VEGFR2D3） fused to human IgG1. Methods： The optimized VEGFRID2/R2D3 gene was cloned into eukaryotie expression vector plRES2-EGFP-Fc, and then the construct named as plRES2-EGFP-VEGF-Trap2 was transfect ed into CHO-K1 cells and screened with antibiotics G418. Protein VEGF-Trap2 was purified by affinity chromatog raphy and the affinity between VEGF-Trap2 with hVEGF165 was evaluated through non-competitive ELISA and bio layer interferometry. Results： DNA sequencing proved that the sequence of eukaryotic expression vector plRES2- EGFP-VEGF-Trap2 was correct, and the CHO-K1 cell line expressing VEGF-Trap2 was got. The functional affini- ty of VEGF-Trap2 1.86×10^7 L/mol was determined by non-competitive ELISA, and the equilibrium dissociation constant of VEGF-Trap2 for hVEGF165 3.13x10^-9 mol/L was analyzed by biolayer interferometry. Conclusion： Re combinant protein VEGF-Trap2 has high affinity with hVEGF165, which suggests that VEGF-Trap2 can be used to block VEGF signaling pathway and provides basis for further study in vitro and in vivo.