【摘要】 重组EPO细胞灌注法培养的收获液,用Q SepharoseXL,C4反相疏水和Superdex75层析柱纯化表达的EPO.经SDS PAGE、PAGE和HPLC分析纯度达98%以上,总回收率为17%.经SDS PAGE分析其表观分子量为32～40kD.用IEF方法测得pI为3.75～4.15.网织红细胞计数法测得体内比活大于1.5×105IU,用ELISA测得体外活性为1.5×106～1.6×106IU.纯化的EPO的氨基酸组成和其它一些性质也进行了研究.更多还原

【Abstract】 Gene-engineering CHO-cells with the capacity to express recombinant human erythropoietin (rhEPO) were cultured in a perfusion mode. The rhEPO from culture supernatent was isolated and purified by a three-step purification process:Q Sephorose XL anion exchange chromatography-C4 reverse-phase chromatography and Superdex 75 molecular sieve chromatography. The purified rhEPO was showed to be homogeneous with a single protein band or a single peak by SDS-PAGE under reducing or non-reducing conditions, PAGE and HPLC. pI 3.75～4.15 of rhEPO was tested by polyacrylamide gel isoelectric focusing. The apparent molecular weight of rhEPO was identified to be 32～40kD by SDS-PAGE under reducing or non-reducing conditions. The western blot result revealed that rhEPO possessed the antigen of native erythropoietin. The specific activity of rhEPO in vitro was estimated 1.5×10~6～1.6×10~6IU by ELISA and more than 1.5×10~5 IU in vivo by Reticulocyte count method.更多还原