人血管抑素 Kringles 1-3 的表达纯化及生物学活性研究

中国生物工程杂志

2011, Vol. 31

Issue (01): 24-28

研究报告

人血管抑素 Kringles 1-3 的表达纯化及生物学活性研究

李壮林¹, 姚雪静², 原桂勇¹

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

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

LI Zhuang-lin¹, YAO Xue-jing², YUAN Gui-yong¹

1. Shandong Simcere-Medgenn Bio-Pharmaceutical Co., Ltd., Yantai 264006, China;
2. Yantai Rongchang Bioengineering Co., Ltd., Yantai 264006, China

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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Kringles 1-3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1.86 μg/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Kringles 1-3治疗肿瘤奠定了初步实验基础。

关键词： 人血管抑素; 表达; 纯化; 活性研究; 毕氏酵母

Abstract:

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.

Key words: Human angiostatin Expression Purification Bioactivity Pichia pastoris

收稿日期: 2010-08-11 出版日期: 2011-01-25

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引用本文:

李壮林, 姚雪静, 原桂勇. 人血管抑素 Kringles 1-3 的表达纯化及生物学活性研究[J]. 中国生物工程杂志, 2011, 31(01): 24-28.

LI Zhuang-lin, YAO Xue-jing, YUAN Gui-yong. Expression, Purification and Bioactivity Research of Human Angiostatin Kringles 1-3. China Biotechnology, 2011, 31(01): 24-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I01/24

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