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The stable Expression of VEGF165 in Mammalian Cells
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- Author:
- No author available
- Journal Title:
- JOURNAL OF CHINESE PHYSICIAN
- Issue:
- 7
- DOI:
- 10.3760/cma.j.issn.1008-1372.2004.07.002
- Key Word:
- VEGF165;CHO细胞;转基因;基因表达
Abstract： 目的探讨pcDNA3.1-VEGF165质粒对哺乳动物细胞的表达和转染.方法将pcDNA3.1-VEGF165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、Westen-blot在转录水平和蛋白质水平上检测VEGF165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VEGF165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600bp的特异性泳带,测序结果与VEGF165 mRNA一致.ELISA显示细胞培养上清的VEGF浓度约为10～20ng/ml.WESTEN-BLOT检测到大小为23KD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-VEGF165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VEGF165蛋白.
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