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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。
Objective To express human vascular endothelial growth factor receptor (VEGFR) -Fc in Chinese hamster ovary cell line DG44 and test its biological activity. Methods The VEGFR1D2 gene of human VEGFR1 and the third immunoglobulin domain (VEGFR2D3) of human VEGFR2 were chemically synthesized. VEGFR1D2-VEGFR2D3 and human IgG1Fc were spliced to form VEGFR by overlapping PCR (Overlap PCR) -Fc fusion gene was inserted into eukaryotic expression vector pD2 to construct recombinant expression plasmid pD2-VEGFR-Fc. The recombinant plasmid pD2-VEGFR-Fc was transfected into DG44 cells with FreeStyleTMMAX Reagent and OptiPROTMSFM. Cells stably expressing VEGFR-Fc protein The expression of VEGFR-Fc in cell culture supernatant was detected by SDS-PAGE, Western blot and ELISA. The expressed VEGFR-Fc protein was purified by HiTrapTMProteinA FF column and its biological activity was detected by microscope and ECV304 model of vascular endothelial cells. Results The size of the amplified product of VEGFR-Fc gene was 1 377 bp. The recombinant plasmid pD2-VEGFR-Fc was confirmed by double enzyme digestion and sequencing. The recombinant plasmid pD2-VEGFR-Fc transfected DG44 cell culture supernatant containing VEGFR- The expression level of Fc was 0.5 g / L. After purified by HiTrapTMProteinA FF, the hybrid protein was removed well. The purified VEGFR-Fc could specifically bind VEGF and inhibit the growth of ECV304. Conclusion The successful expression of VEGFR-Fc with biological activity in DG44 cells lays the foundation for further study on its role in inhibiting angiogenesis and anti-tumor.