人血管内皮生长因子受体-Fc在DG44细胞中的表达及其生物活性

目的在中国仓鼠卵巢细胞DG44中表达人血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)-Fc,并检测其生物活性。方法化学合成人VEGFR1的第2免疫球蛋白结构域(VEGFR1D2)基因和人VEGFR2的第3免疫球蛋白结构域(VEGFR2D3)基因,通过重叠PCR(Overlap PCR)将VEGFR1D2-VEGFR2D3和人IgG1Fc拼接形成VEGFR-Fc融合基因,插入真核表达载体pD2中,构建重组表达质粒pD2-VEGFR-Fc,在FreeStyleTMMAX Reagent和OptiPROTMSFM介导下转染至DG44细胞中,MTX加压筛选稳定表达VEGFR-Fc蛋白的细胞株,SDS-PAGE、Western blot和ELISA法检测细胞培养上清中VEGFR-Fc蛋白的表达。表达的VEGFR-Fc蛋白经HiTrapTMProteinA FF柱纯化后,利用显微镜观察法和血管内皮细胞ECV304模型检测其生物活性。结果 VEGFR-Fc基因扩增产物大小为1 377 bp;重组表达质粒pD2-VEGFR-Fc经双酶切和测序证明构建正确;质粒pD2-VEGFR-Fc转染的DG44细胞培养上清中含VEGFR-Fc蛋白,表达量为0.5 g/L;细胞培养上清经HiTrapTMProteinA FF柱纯化后,杂蛋白去除效果较好;纯化的VEGFR-Fc能与VEGF特异性结合,抑制ECV304生长。结论成功在DG44细胞中表达了具有生物活性的VEGFR-Fc,为进一步研究其在抑制血管生成和抗肿瘤中的作用奠定了基础。

Expression of human vascular endothelial growth factor receptor-Fc in DG44 cells and biological activity of expressed product

Objective To express human vascular endothelial growth factor receptor(VEGFR)-Fc in Chinese hamster ovary(CHO)DG44 cells and determine its biological activity.Methods The sequence VEGFR1D2-VEGFR2D3 encoding the second and the third human immunoglobulin domains of VEGFR2 was synthesized,and fused with human IgG1Fc by overlap PCR to generate VEGFR-Fc fusion gene,which was then subcloned into eukaryotic expression plasmid pD2.The constructed recombinant plasmid pD2-VEGFR-Fc was transfected to DG44 cells in mediation of FreeStyleTM MAX Reagent and OptiPROTM SFM,and a cell line stably expressing VEGFR-Fc protein was screened by MTX pressure screening.The expressed VEGFR-Fc in cell culture supernatant was determined by SDS-PAGE,Western blot and ELISA,then purified through HiTrapTM ProteinA FF column,and determined for activity by using microscopy and endothelial ECV304 cell model.Results The length of PCR product of VEGFR-Fc gene was 1 377 bp.Restriction analysis and sequencing proved that recombinant plasmid pD2-VEGFR-Fc was constructed correctly.VEGFR-Fc protein was detected in culture supernatant of DG44 cells transfected with plasmid pD2-VEGFR-Fc,of which the expression level was 0.5 g / L.After purification by HiTrapTM ProteinA FF column chromatography,the foreign protein in cell culture supernatant was removed effectively.Purified VEGFR-Fc showed specific binding to VEGF and inhibited the growth of ECV304 cells.Conclusion The VEGFR-Fc with biological activity was successfully expressed in DG44 cells,which laid a foundation of further study on its role in angiogenesis inhibition and anticancer therapy.