两步柱色谱法纯化CHO表达的重组人β神经生长因子(β-rhNGF)

Two-step Purification of Recombinant Human Beta Nerve Growth Factor(β-rhNGF) Secreted by CHO Cells with Chromatographic Method

基因重组技术生产蛋白药物较传统提取生产方式具有诸多优点。运用一步离子交换色谱层析(Sepharose Fast Flow)和反相(C4)色谱层析串联纯化了CHO工程细胞株分泌表达的重组人β神经生长因子(β-rhNGF),纯化产物经SDS-PAGE及反相HPLC分析纯度均达到95%以上,生物学活性经PC12细胞和鸡胚背根神经节测定均与Sigma标准品无差异。产物经两步纯化后的收率达70%以上,为建立该产品切实可行的工业化纯化工艺提供了依据。

Nerve growth factor （NGF） was firstly discovered as a member of neurotrophin family, and the research and development of NGF has been lasting more than fifty years since its discovery. To this end, a twostep and high - yield chromatographic method which consists of cation ion-exchange chromatography and reversed-phase chromatography was reported to isolate recombinant human beta nerve growth factor （β- NGF） secreted by constructed Chinese hamster ovary cells （CHO/dhfr-） from the culture media. Through the process of purification, the purity of protein which was determined by SDS-PAGE and RP-HPLC has reached to 95% , and the recovery of β- NGF routed by RP-HPLC could be 70%. Furthermore, the biological activity of final purified protein evaluated by PC12 cells and dorsal root ganglia （DRG） exhibited the same performance as the standard protein of β-NGF bought from Sigma, which indicated that there is no loss of biological activity through the isolation process. The conclusion suggested that an economical isolation method of recombinant human β- NGF could be practiced on the industrial process of purification.