摘要
目的 :探索原核基因工程产品质量控制的指标。方法 :以重组粒细胞集落刺激因子(G CSF)为模型 ,将其中构象不均一组分进行分离 ,并对每个成分进行鉴定。结果 :重组人G CSF在大肠杆菌中以包涵体形式表达 ,经纯化和复性后 ,SDS PAGE鉴定为单一蛋白带 ,但在非变性PAGE中出现 3条带 ,继而采用制备型非变性PAGE成功地将 3种成分分离。 3种成分在同样的缓冲体系中分别存放 3个月 ,仍未见互变异构现象。经激光解析飞行时间质谱检测 ,3种成分相对分子质量相同 ,测序表明 ,N端连续的 5个氨基酸残基排列完全相同。结论 :3种成分为重组蛋白的不均一折叠形成的构象异构体。
Objective:To investigate the role of conformational heterogeneity in quality control of genetic engineering drugs.Methods:Components of rhG CSF were separated.Each component was characterized.Results: Three bands were observed in native PAGE of renatured purified rhG CSF which was expressed in E.coli as inclusion bodies and showed one single band in SDS PAGE.Using preparative native PAGE, they were successfully separated. No interchange among them was found even after 3 months under the same buffer system.MALDI TOF and N terminal sequencing showed the same molecular weight and amino acid sequence.Conclusions:The results suggest that they have the same primary structure but different higher order structures. [
出处
《军事医学科学院院刊》
CSCD
北大核心
2000年第4期241-243,共3页
Bulletin of the Academy of Military Medical Sciences
基金
全军医药卫生科研基金!课题 (96Q37)
