摘要
用无血清培养基培养分泌表达rhEPO-Fc融合蛋白的工程细胞株MY06(CHO细胞),收集发酵培养上清,通过离心、过滤、亲和层析、离子交换层析、分子筛等方法对目的蛋白进行纯化,通过lowry法、SDS-PAGE、Western-blot、HPLC、ELISA及IEF等方法研究目的蛋白质量,以建立rhEPO-Fc融合蛋白的发酵、纯化工艺,并探寻该融合蛋白的质量检测方法。通过该工艺,rhEPO-Fc蛋白表达量高达2g/L,蛋白纯化得率达45%以上,纯度可达98%以上,相对分子质量约为60kDa,t1/2长达38h,免疫印迹证明具有天然EPO的免疫原性。研究结果表明该生产工艺可获得rhEPO-Fc的高效表达,纯化得率高,质量检验方法稳定可靠,适用于大规模生产。
To explore the technics for high expression, purification, quality control of recombinant human erythropoietin and IgG Fc fusion protein, rhEPO-Fc fusion protein expressed by CHO cells in serum-free medium was collected and purified with a three-step purification process-affinity chromatography, ion exchange chromatography and molecular sieve chromatography; and its quality was controlled by protein concentration, SDS-PAGE,Western-blot,HPLC,ELISA and IEF, et al. The research findings are that the expression output of rhEPO-Fc was highly reached 2g/L, the purify rate was over 45%, its purify was over 98%, its relative molecular weight is about 60kDa, t1/2 in vivo of rhEPO-Fc is over 38h, western blot analysis showed rhEPO-Fc has nature antigenicity. The expression output and purify rate of the produce technics is high, quality inspection methods are stability and reliable, which could be fit for in large-scale production of rhEPO-Fc.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第6期6-9,共4页
China Biotechnology
基金
上海市创新基金资助项目(0602H1203)
关键词
重组人促红细胞生成素
免疫球蛋白Fc段
融合蛋白
发酵纯化工艺
质量研究
Recombinant huma The technics of fermentor and purify n erythropoietin Fc segment of immunoglo- bulin G Fusion protein Quality control
