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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 contains four kringle domains and is a potent angiogenic inhibitor. The gene of human angiostatin Kringles1-3 was amplified from human embryonic liver cDNA library by PCR and recombined into pPIC9K vector to obtain the recombinant vector pPIC9K-K1-3. Then, the recombinant plasmid was transformed into Pichia pastoris GS115. The K1-3 protein was 41.2 mg / L in a 5 L fermentor. The supernatant of the fermentation broth was concentrated, dialyzed and then purified on a Lysine Sepharose 4B column. The K1-3 protein was more than 98% pure and purified Rate of more than 95%. K1-3 significantly inhibited the migration of bFGF-stimulated human microvascular endothelial cells, with a protein concentration (IC50) of 1.86 μg / ml required to achieve a 50% inhibitory effect. K1-3 also inhibited chick chorioallantoic membrane vascular growth, the inhibition rate of 95%. For the application of human angiostatin Kringles1-3 treatment of the tumor has laid a preliminary experimental basis.