Abstract
Erythropoietin (EPO) genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHO-K12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 106 cells were obtained at 400 μg/mL G418. Based on the C10 cell, another vector pHY/dhfr (dihydrofolate reductase) that carries a dhfr gene and a selecting marker of hygromycin B resistant gene was transferred to this cell. Several cell clones were obtained at 200 μg/mL hygromycin B. These cell clones that can express both EPO gene and exogenous dhfr gene were selected under the progressively increased concentration to 1 μmol methotrexate (MTX). Some high EPO expression cell clones were obtained, the highest expression was 2 400 IU/d in 106 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using un-dhfr negative cell was primarily established. EPO bioactivity was found by using TF-1 cell.
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