红细胞生成素cDNA在中国仓鼠卵巢细胞中的稳定表达

Stable Expression of Erythropoietin cDNA in Chinese Hamster Ovary Cells

将(?)红细胞生成素(EPO)cDNA构建的重组表达质粒用电穿孔法引入COS-7细胞,ELISA和红系集落测活结果表明,该重组质粒在哺乳动物细胞中能够表达有生物活性的红细胞生成素。进一步将其转染CHO-dhfr^-细胞,经氨甲喋呤(MTX)加压扩增,混合细胞中各克隆表达水平比较一致,细胞的平均表达水平为2-3μg/10~6 Cells/24hr。细胞冻存后复苏其表达水平与冻存前一致,表明外源基因整合稳定。

The recombinant erythropoietin expression plasmid.constructed by inserting erythropoietin cDNA in cloning sile of eukaryotic expression vector pCDS,was introduced into COS-7cells,ELISA and colony formation assay indicated that biologically active erthropoietin was expressed. The plasmid was then introduced into CHO-dhfr cells b> means of electroporation and the copy number of foreign gene was amplified by exposing to the culture media containing gradually increasing concentration of methetrexate (MTX). After amplification with 2×10-7mol/L MTX.the average expression of mixed cells reached 2-3μg/106cells/24hrs and the expression levels of different clones varied little. After 3 months of cryopreservation of the cells,the secretion of erythropoietin didn't seem to decrease,which suggested that the foreign target gene had stably integrated into the genome of host cells.