层析法去除重组人血清白蛋白干扰素α2b融合蛋白的内毒素  

Removing Endotoxin from rHSA-IFNα2b by Chromatography

作　　者：徐向荣[1] 马国昌[2] 王昌梅[3] 单剑峰[3] 沈玲[3] 黄岩山[3] 周金宝[3] 

机构地区：[1]浙江大学医学院附属妇产科医院,杭州310006 [2]浙江大学药学院,杭州310031 [3]杭州九源基因工程有限公司,杭州310018

出　　处：《生物工程学报》2008年第1期159-163,共5页Chinese Journal of Biotechnology

摘　　要：用柱层析方法在生产重组人血清白蛋白干扰素a2b融合蛋白过程中去除产品中的内毒素。所用柱层析组合为Blue-sepharose亲和柱层析、SOURCE 15 ISO疏水柱层析、Q Sepharose F.F.离子交换柱层析、Sephadex G25 Coarse凝胶过滤柱。采用鲎试剂法检测柱层析各阶段得到蛋白中的内毒素含量;并用RP-HPLC方法测定柱层析各阶段得到蛋白的纯度与浓度,求出每毫克蛋白的内毒素含量。结果为每步柱层析过程式均有去除内毒素的作用,最终所得蛋白的内毒素含量降为1 EU/mg,去除率达到99.9%。因而生产重组人血清白蛋白干扰素a2b融合蛋白所用分离纯化柱层析技术在纯化蛋白的同时能有效的去除产品中内毒素,所得产品内毒素的含量远低于药典对注射剂的内毒素含量要求。We used chromatography to remove endotoxin during the purification of rHSA-IFNα2b （recombinant human serum al- bumin-Interferon alpha 2b, rHSA- IFNα2b）. Affinity chromatography of Blue-sepharose, hydrophobic interaction chromatography of SOURCE 15 ISO, ion exchange chromatography of Q Sepharose Fast Flow and gel filtration of sephadex G25 Coarse were applied consequently. The endotoxin levels were measured by Limulus Amebocyte Lysate gel-clot assay. Protein purity and concentration were determined by RP-HPLC. Up to 99.9% endotoxin of rHSA-IFNα2b was removed and to a concentration of 1 EU/mg. This process was effective to purify rHSA-IFNα2b and remove exdotoxin simultaneously.

关 键 词：人血清白蛋白 干扰素Α2B 柱层析 内毒素 

分 类 号：Q789[生物学—分子生物学]