聚乙二醇定点修饰重组人粒细胞集落刺激因子突变体的研究

Study of Target Pegylated Recombinant Mutant Human Granulocyte Colony Stimulating Factor

摘要:

研究了重组人粒细胞集落刺激因子突变体（rmhG-CSF）的聚乙二醇化修饰、分离纯化和活性鉴定。通过对人重组粒细胞集落刺激因子（rhG-CSF）第1，3，4，5，17位氨基酸进行突变，并在C末端加了一个半胱氨酸，获得了体外活性为原型rhG-CSF 150％以上的重组人粒细胞集落刺激因子突变体（rmhG-CSF）。然后用分子量为20kD的甲氧聚乙二醇马来酸酐（mPEG-Mal）修饰rmhG-CSF，反应混合物经离子交换和凝胶过滤柱纯化，得到纯化的聚乙二醇重组人粒细胞集落刺激因子突变体（PEG-rmhG-CSF）。SDS-PAGE电泳分析表明纯化后的PEG-rmhG-CSF的纯度大于95％，体外活性分析表明PEG-rmhG-CSF活性优于目前临床使用的聚乙二醇重组人粒细胞集落刺激因子（PEG-rhG-CSF，Neulasta^?），药代动力学研究表明PEG-rmhG-CSF体内半衰期约为14h，比修饰前延长了7倍。

Abstract:

Recombinant mutant human granulocyte colony stimulating factor (rmhG-CSF) was pegylated, purified and characterized. rhG-CSF was mutated in position 1，3，4，5，17,and cysteine was added in C-terminal. rmhG-CSF was pegylated by PEG-Mal 20000 and separated by ion-exchange chromatography, gel filtration chromatography. Analysis of SDS-PAGE showed thar the purity of the separated PEG-rmhG-CSF was greater than 95%. and in intro and in vivo bioactivity study showed that target modified PEG-rmhG-CSF kept full bioactivity which was better than traditional pegylation method, and longer half-life was proved in mice.