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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。
The cDNA of human granulocyte-macrophage colony-stimulating factor (GM-CSF) was modified by polymerase chain reaction (PCR) and synthetic oligo DNA primers. Change some G or C to A or T without changing the amino acids to avoid the formation of unfavorable secondary structures and to replace some of the codons with codons that are of interest to E. coli. The modified GMCSF cDNA expressed higher in E. coli than its parent strain. SDS-polyacrylamide gel electrophoresis showed that the expression level accounted for about 20% of the total bacterial protein, and the immunological and biological activity tests showed that the expressed product possessed the conformation and biological activity of native GM-CSF. Partially purified recombinant human GM-CSF has been used to maintain the growth of the human GM-CSF-dependent TF-1 cell line.