人血管抑素Kringles 1-3的表达纯化及生物学活性研究  

Expression,Purification and Bioactivity Research of Human Angiostatin Kringles 1-3

作　　者：李壮林 姚雪静 原桂勇 

机构地区：[1]山东先声麦得津生物制药有限公司,烟台264006 [2]烟台荣昌生物工程有限公司,烟台264006

出　　处：《中国生物工程杂志》2011年第1期24-28,共5页China Biotechnology

摘　　要：人血管抑素包含了4个环饼状结构域(Kringle),是一个有效的血管生成抑制因子。采用PCR方法从人胚肝cDNA文库中扩增了人血管抑素Kringles1-3的基因,重组于pPIC9K载体中,获得重组载体pPIC9K-K1-3,然后转化毕赤酵母细胞GS115,获得重组菌株。K1-3蛋白在5 L发酵罐的表达量达41.2 mg/L,发酵液上清经过浓缩、透析,然后用Lysine Sepharose 4B层析柱纯化,得到的K1-3蛋白纯度大于98%,纯化回收率达到95%以上。K1-3能明显抑制bFGF刺激的人微血管内皮细胞的迁移,达到抑制效果为50%时所需的蛋白浓度(IC50)为1.86μg/ml。K1-3也能抑制鸡胚绒毛尿囊膜血管的生长,抑制率达95%。为应用人血管抑素Kringles1-3治疗肿瘤奠定了初步实验基础。Human angiostatin is composed of 1-4 kringles of plasminogen,which can inhibit the angiogenesis of tumor,the tumor growth and metastasis through preventing tumor′s blood and nutrition from supplying.The complete encoding cDNA of human angiostatin Kringles 1-3 was isolated from human embryo liver with PCR and inserted to pPIC9K vector.Then,K1-3 was expressed in secretory P.pastoris expression system by induction of methanol and the expression yield of K1-3 in 5 L fermenter was 41.2 mg/L.The supernatant of cultivation liquid was collected,then concentrated and dialyzed.Recombinant human K1-3 was purified by affinity chromatography with Lysine Sepharose 4B.As a result,HPLC showed purity of K1-3 was more than 98% and the recovery yield was more than 95%.Recombinant human K1-3 inhibited significantly the migration of human microvascular endothelial cells and half inhibitory concentration was 1.86μg/ml.Research showed that K1-3 suppressed specifically the angiogenesis on the CAMs with 95% inhibition.

关 键 词：人血管抑素 表达 纯化 活性研究 毕氏酵母 

分 类 号：Q789[生物学—分子生物学]