人血管抑素在毕氏酵母中的表达及其活性测定  

Expression of Human Angiostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity

作　　者：辛利[1] 张励[2] 徐韧 张倩[1] 叶勤[2] 李载平[1] 甘人宝[1] 

机构地区：[1]中国科学院上海生命科学研究院 [2]华东理工大学生物反应器国家重点实验室,上海200237

出　　处：《生物化学与生物物理学报》2001年第3期291-295,共5页

基　　金：中国科学院上海生命科学研究院资助项目&&

摘　　要：利用RT PCR方法从人肝细胞癌HepG2细胞株中扩增得到编码人血管抑素 (angiostatin)的cDNA。将其插入毕氏酵母表达载体 pPIC9K中 ,转化毕氏酵母 ,获得了整合多拷贝血管抑素基因的菌株。发酵液经赖氨酸亲和柱纯化所得到的重组蛋白的产量为 2 5mg/L。蛋白质氨基酸序列分析结果与国外报道一致。重组蛋白特异地抑制bFGF刺激的牛主动脉内皮细胞的增殖 ,ED50 约为 3mg/L。Human angiostatin cDNA was amplified from human hepatoma cell line HepG2 using RT PCR and was cloned into pPIC9K vector. Recombinant Pichia pastoris strain with 4 copies of angiostatin gene was obtained. Recombinant protein was purified by lysine affinity Sepharose column and the finally purified angiostatin was 25 mg/L, higher than previously reported 17 mg/L. Amino acid sequence analysis revealed the identity of our protein the same with that previously reported. Recombinant angiostatin inhibited specifically the proliferation of bovine aortic endothelial cell stimulated by bFGF, with ED 50 being about 3 mg/L.

关 键 词：血管抑素 多拷贝整合 华氏酵母 抑制作用 内皮细胞 基因工程表达 

分 类 号：Q279[生物学—细胞生物学]