【摘要】 为构建重组人红细胞生成素(recombinant human erythropoietin,rhEPO)二聚体真核表达载体,应用PCR方法扩增EPO cDNA,PCR产物克隆入T载体后,经酶切、连接、转化等过程分别构建了3个EPO二聚体的真核表达载体pBT-1c、pBT-2s及pBT-3c,经测序序列完全正确。然后将3个真核表达载体分别转染于CCS-7细胞及CHO-dhfr-细胞中,用ELISA方法检测,它们在COS-7的瞬时表达量分别为4IU／ml、11.5IU／ml和7.2IU／ml。其中EPO二聚体真核表达载体pBTsv稳定转染CHO-dhfr-细胞后,用氨甲喋呤(MTX)逐渐加压的方法筛选到阳性克隆,表达量可达到4000IU／106cells／72h。更多还原

【Abstract】 To construct recombinant human erythropoietin dimmer eukaryotic expression vector, EPO gene was generated by Polymerase Chain Reaction (PCR )from cDNA, the PCR prouducts were cloned to pGEM-T vector and the DNA was digested with NheⅠ/ HindⅢ or HindⅢ/ XhoⅠ to obtain 1.2 kb fragments. Then the fragments were subcloned into eukaryotic expression vector to generate plasmid pBT-1c ( 6.2kb ), pBT-2s ( 6.0kb ) and pBT-3c (7.3kb ), respectively. The sequences were confirmed completely by sequencing analysis. All the three plasmids were transfected to COS-7 cell and the EPO dimmer expression was detected by Enzyme Linked Immunosorbent Assay (ELISA) . The expression level was 4IU/ml, 11.5 IU/ml and 7.2IU/ml respectively. The pBT-2s plasmid was transfected to CHO-dhfr- cell and the positive clone was selected by increasing the methotrexate (MTX) . The expression level of EPO dimmer was up to 4 000IU/106cell/72h.更多还原