论文部分内容阅读
临床用重组人促红细胞生成素(rhEpo)是中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)表达的糖蛋白,糖基对稳定蛋白的结构和生物活性非常重要,但CHO表达体系生产成本高、产量低.以大肠杆菌表达的促红细胞生成素为非糖基化蛋白(rh-ngEpo),对其进行聚乙二醇(PEG)修饰可以提高蛋白稳定性和体内循环半衰期.本文采用分子量为20000的N-末端专一性的单甲氧基聚乙二醇-丙醛(mPEG-ALD)修饰rh-ngEpo,对影响修饰反应的因素进行了考察.结果表明,在最佳反应条件下,单修饰率可达55%.修饰混合物经离子交换层析分离,获得了纯度大于95%的单修饰产物,其二、三级结构证明与原蛋白相似.肽图分析结果表明,PEG绝大部分修饰在蛋白N-末端的氨基酸残基上.ELISA分析表明,单修饰产物的体外活性虽然比修饰前减少30%,但热稳定性得到显著增强,在SD大鼠体内的药代动力学性质得到显著提高.研究结果表明,PEG可以在一定程度上替代糖基的作用,PEG修饰的非糖基化Epo有望成为一种新型的促红细胞生成蛋白药物.
Clinical recombinant human erythropoietin (rhEpo) is a glycoprotein expressed in Chinese hamster ovary cells (CHO). The glycosylation is very important for the structure and biological activity of the stable protein. However, the production cost of the CHO expression system is high, Low yield.The E.coli-expressed erythropoietin as a non-glycosylated protein (rh-ngEpo), its polyethylene glycol (PEG) modification can improve protein stability and circulatory half-life.In this paper, the molecular weight of 20000 (MPEG-ALD) modified rh-ngEpo was used to investigate the factors that affect the modification reaction.The results showed that under the optimum reaction conditions, single The modification rate was up to 55% .The modified mixture was separated by ion-exchange chromatography to obtain a single-modified product with a purity greater than 95%, and the secondary and tertiary structures were similar to the original protein.The peptide analysis showed that most of PEG At amino acid residues at the N-terminus of the protein, ELISA analysis showed that although the in vitro activity of the monodisperse product was reduced by 30% compared with that before modification, the thermal stability was significantly enhanced and the pharmacokinetic properties were significantly improved in SD rats Improve Results show, PEG can replace the role of sugar moieties to some extent, PEG-modified non-glycosylated Epo is expected to become a new type of protein drug erythropoietin.