无血清培养基生产重组人红细胞生成素的纯化

Study of Purification of Recombinant Human Erythropoietin Produced with FBS Free Medium .

用无血清培养基在生物反应器中培养CHO EPOC2细胞株 ,培养上清中重组人红细胞生成素表达水平达 1 0～ 2 1mg/L .培养上清经过三步纯化后纯度可达到 98%以上 ,比活性约为 1 5×1 0 5U/mg.纯化第一步使用反相柱层析 ,可将样品体积浓缩约 30倍 .取其收集液进行DEAE 离子交换柱层析 ,最后进行分子筛层析 ,全过程回收率为 4 0 %左右 .SDS PAGE表明 ,所制备终产品分子质量为 35～ 4 0ku ,等电聚焦方法测其等电点在 3 75～ 4 1 5之间 ,均属文献报道范围 ;ELISA和蛋白质印迹实验结果证明其具有天然红细胞生成素抗原性 ;采用溴化氰裂解方法做肽谱电泳 ,结果与理论推测相符 .该纯化路线简单、迅速、高效 ,重复性好 。

CHO EPO C2 cells were cultured in the Packed Bed Bioreactor with free fetal bovine serum medium, and the supertant contained 2 000～3 000 U/ml of recombinant Human EPO(rHuEPO). The SDS PAGE result of the final products from the reported purification schemes was a single band, which purity was over 98% with measurement of UV scanning. The specific activity was 1 5×10 5 U/mg protein. Reserch showed that the molecular weight was about 35～40 ku and the pI was about 3 75～4 15.The product possessed the antigenity of native HuEPO. The peptide electrophoresis result was in accord with theory deducation. 15 amino acid sequence of rHuEPO N terminal was as the same as native HuEPO. All these results described above revealed that the final product were high purity rHuEPO and the purification procedure was rapid and efficient, which can be used to produce clinic rHuEPO in large scale.