VEGF_(121)在哺乳动物细胞中的稳定表达

Transfection expression of VEGF_(121) in mammalian cells

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 transfection and expression of VEGF121 in mammalian cells. Methods: pcDNA3.1-VEGF121 plasmid were transfected into CHO cells by electroporation and clones were screened by G418. The transcription and expression of VEGF121 in CHO cells were tested by RT-PCR, ELISA and Western-blot. The biologic activities of VEGF121 secreted by CHO cells were investigated by Mile′s assay and endothelial cells MTT assay. Results: We Obtained anti-G418 CHO cells mono-clones. RT-PCR, ELISA and Western-blot proved that there was transcription and expression of VEGF121 in anti-G418 CHO cells. The expressed VEGF121 could increase the vessel permeability and promote the proliferance of endothelial cells. Conclusions: pcDNA3.1-VEGF121 plasmid can express active VEGF121 well in mammalian cells.