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

作　　者：黄岩山[1] 金荣[2] 刘晓妮[2] 王昌梅[2] 戎亚雯 陈智[1] 

机构地区：[1]浙江大学医学院附属第一医院传染病研究所,杭州310003 [2]杭州九源基因工程有限公司,杭州310018

出　　处：《生物工程学报》2007年第5期919-923,共5页Chinese Journal of Biotechnology

基　　金：浙江省重大科技攻关项目(No.2005C13025)~~

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-CSF150%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,NeulastaR○),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。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.

关 键 词：聚乙二醇化修饰 重组人粒细胞集落刺激因子突变体 纯化 鉴定 

分 类 号：Q78[生物学—分子生物学]