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目的探讨pcDNA3.1-VEGF121质粒对哺乳动物细胞的表达和转染。方法将pcDNA3.1-VEGF121质粒用电转的方法导入CHO细胞中,G418筛选细胞克隆;通过RT-PCR,ELISA,Western-blot在转录水平和蛋白质水平上检测VEGF121在转基因CHO细胞中的表达;通过内皮细胞增殖实验和血管通透性实验检测所表达的VEGF121的生物学活性。结果获得有G418抗性的CHO细胞克隆;RT-PCR扩增出一条大小约450bp的特异性泳带,测序结果与VEGF121mRNA一致;ELISA显示细胞培养上清的VEGF浓度约为8～22ng/ml;Western-Blot检测到大小为17KD的特异性蛋白质条带;内皮细胞增殖实验显示细胞培养上清具有促内皮细胞增殖作用;血管通透性实验显示细胞培养上清明显增加毛细血管通透性。结论pcDNA3.1-VEGF121质粒真核表达系统能在哺乳动物细胞中表达具有良好生物学活性的VEGF121蛋白。
Objective To investigate the expression and transfection of pcDNA3.1-VEGF121 plasmid in mammalian cells. Methods The pcDNA3.1-VEGF121 plasmid was transfected into CHO cells by electroporation, and the cell clones were screened by G418. The expression of VEGF121 in transgenic CHO cells was detected by RT-PCR, ELISA and Western-blot at transcriptional and protein levels. The biological activity of VEGF121 expressed was examined by endothelial cell proliferation assay and vascular permeability assay. Results A G418-resistant CHO cell clone was obtained. A specific 450 bp band was amplified by RT-PCR. The sequencing result was consistent with that of VEGF121 mRNA. ELISA showed that the concentration of VEGF in the cell culture supernatant was about 8-22 ng / ml. The specific protein band of 17KD was detected by Western-Blot. The endothelial cell proliferation assay showed that the cell culture supernatant had the function of promoting endothelial cell proliferation. The vascular permeability test showed that the cell culture supernatant obviously increased the capillary permeability. Conclusion The pcDNA3.1-VEGF121 plasmid eukaryotic expression system can express VEGF121 protein with good biological activity in mammalian cells.