论文部分内容阅读
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。
To construct eukaryotic expression vector of recombinant human erythropoietin (rhEPO) dimer, EPO cDNA was amplified by PCR and cloned into T vector. After digestion, ligation and transformation, The eukaryotic expression vectors pBT-1c, pBT-2s and pBT-3c of 3 EPO dimers were completely correct. Then the three eukaryotic expression vectors were transfected into CCS-7 cells and CHO-dhfr- cells respectively and detected by ELISA. The transient expression of COS-7 in these cells were 4IU / ml, 11.5IU / ml and 7.2 IU / ml. The EPO dimer eukaryotic expression vector pBTsv stably transfected CHO-dhfr- cells, with methotrexate (MTX) gradually pressurized method to screen positive clones, the expression amount can reach 4000IU / 106cells / 72h.