pBV—IL—6重组质粒的构建及其在E.coli细胞中的超高表达

本研究通过计算机分析设计、人工合成的寡核苷酸引物进行定点诱变和转译起始区优化,再经 PCR 扩增和体外重组,获得重组质粒 pBV-IL-6,转化大肠杆菌获得重组人白细胞介素6的工程菌 EcoliJM103/pBV-IL-6.结果,表达 IL-6占菌体总蛋白的71%.经 IL-6依赖的小鼠杂交瘤细胞系7TDI 和~(?)H-TdR 掺入法测定表达产物粗提物的 IL-6生物活性为10~6U/L,经凝胶过滤纯化与复性之后 IL-6比活性为10~(?)U/mg.表达产物为缺失 N 端25个氨基酸的人白细胞介素6.

Construction of recombinant interleukin-6 plasmid pBV-IL-6 and its overexpression in E.coli

In this paper,by means of PCR amplification,site-specific mutagenesis,DNA recombination in vitro and optimization of translation initiation,we constructed a recombinant plasmid pBV-IL-6 that overproduces biologically active human interleukin-6(rhIL-6)in E.coli.The expressed rhIL-6 lacks the first 25 amino acids of mature hIL-6 and accounts for 71% of the total bacteria proteins.The reasons why a deleted rather than mature IL-6 was chosen for expression in E.coli are as follows:1)The specific bio-activities of the deleted hIL-6 are the same as,even more than that of the mature form according to the published data;2)The deleted part of IL-6 coding region is rich in C-G andeasy to form secondary structure in mRNA,which is not good for expression;3)The re-designed region for initiation site of translation encodes a hydrophilic amino-end which favours highly translation initiation and expression of hIL-6 in E.coli.