摘要
Erythropoietin （EPO） genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHOK12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 10\+6 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 10\+6 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using undhfr negative cell was primarily established. EPO bioactivity was found by using TF1 cell.
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.
基金
ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina (GrantNo .395 0 0 0 84 )
