含凝血酶识别位点的重组人粒细胞/巨噬细胞集落刺激因子在大肠杆菌的表达  

Expression of rhGM-CSF with Thrombin Cleavage Site in E. coli

作　　者：韩金祥[1] 李翠玲[1] 宋长征[1] 梁浩 张翠[1] 王美玲[1] 王革[2] 

机构地区：[1]山东省医学科学院生物技术中心,济南250062 [2]中国科学院生物物理研究所

出　　处：《中国生化药物杂志》1997年第6期282-285,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：采用PCR技术，从细胞中获取粒细胞／巨噬细胞集落刺激因子cDNA，并在5′端设计上凝血酸酶切位点、5个组氨酸密码子、起始密码子及EcoR1酶切位点，在3′端设计上BamH1酶切位点。将扩增产物酶切后克隆到pBV220质粒的EcoRⅠ－BamHⅠ酶切位点，转化大肠杆菌DH5α，筛选阳性重组子（pGM09／DH5α）。在E.coli中获高效表达，表达量占总蛋白的31．2％，生物活性为1．1×107U／L发酵液，纯化工艺简化。The E. coli vector pGM09/DH5α was custracted by cloning the GM-CSF cDNA obtained byPCR. The cDNA had thrombin cleavage site, codons of five histidines, EcoR Ⅰ site at its 5′ end andBamH Ⅰ site at its 3′ end. The pGM09 was transformed into E. coli DH5α and a high expression strainwas obtained. The expressed rhGM-CSF consisted of 31.2% of total protein and its biological activityin ferment liquid was 1.1 ×107 U/L. The technology of purification is simple.

关 键 词：粒细胞 基因表达 GM-CSF CDNA 

分 类 号：R392-33[医药卫生—免疫学] R394.8[医药卫生—基础医学]