摘要
应用聚合酶链反应(PCR)法和人工合成的寡聚DNA引物,对人粒细胞-巨噬细胞集落刺激因子(GM-CSF)的cDNA作了修饰。在不改变氨基酸的前提下,将一些G或C改成A或T,以避免形成不利的二级结构,并将一些密码子换成大肠杆菌喜用的密码子。修饰后的GMCSF cDNA在大肠杆菌的表达水平高于其母株。SDS-聚丙烯酰胺凝胶电泳表明其表达量约占菌体总蛋白的20％,免疫学和生物学活性检测表明该表达产物具有天然GM-CSF的构型和生物学活性。部分纯化的重组人GM-CSF已用于维持依赖人GM-CSF的TF-1细胞系的生长。
Polymerase chain raction ( PCR ) and synthesized oligo-nucleotide pri mers were used to modify the 5' terminal cDNA sequence of human gra nulocyte-macrophage colony stimulating factor. As long as the amino ac-id is unchanged, some G or C have been changed to A or T, to avoid the undesirable secondary structure. Some codons were changed to those pre-ferred by the E.coli system. The expression Ievel of the modified GM-CSF cDNA in E.coli is much higher than its wild type. SDS-PAGE ana-lysis revealed that the GM-CSF accounted for about 20% of total cell protein. ELISA and biological activity assays showed that this recombin-ant protein is similar to the nat鹯ai human GM-CSF.
出处
《病毒学报》
CAS
CSCD
北大核心
1993年第2期136-143,共8页
Chinese Journal of Virology
基金
国家863高科技生物技术领域资助
关键词
粒细胞
巨噬细胞
集落刺激因子
Granulocyte-macrophage colony stimulating factor, Poly-merase chain reaction, Gene modification, Expression in E.coli
