可溶型三聚体血管生长抑制因子Kringle 5的克隆,表达,纯化及活性研究

Cloning,Expression,Purification and its Biological Activity Study of the Soluble Tripolymer Recombinant Angiogenesis Inhibitor Kringle 5

血管生长抑制因子Kringle 5是目前发现的抑制血管内皮细胞增殖和肿瘤生长的活性最强的纤溶酶原片段,特异性高而毒副作用小,在肿瘤的治疗方面具有潜在的巨大价值和广阔的应用前景。根据K5基因的序列设计PCR引物,通过PCR从已有的克隆载体扩增出人纤维蛋白溶酶原的K5部分基因,将K5基因克隆入原核表达载体pET15b,经序列测定,成功构建了pET15b-K5非融合表达载体。将重组载体导入大肠杆菌中IPTG诱导表达,SDS-PAGE分析目的蛋白主要以可溶形式存在于菌体中,破碎后上清通过阳离子交换层析,纯化获得纯度大于95%的目标蛋白,脱盐后对分子量测定推测形成了三聚体。通过鸡胚绒毛尿囊膜法证明蛋白产物对鸡胚绒毛尿囊膜血管增生有一定的抑制作用。

The Kringle 5 domain of plasminogen,which was previously shown to inhibit angiogenesis in vitro and vivo,is one of the most potent angiogenesis inhibitors described to date.The Kringle 5 gene was amplified from pET32a-K5 by PCR,and then was inserted into pET15b to construct a prokaryotic vector pET15b-k5.The target protein was highly expressed in E.coli BL21（DE3） and was mainly present in the precipitation observed by SDS-PAGE.The protein was purified by ion-exchange chromatography（SP Sepharose Fast-Flow） using the NaCl linear gradient strategy.The analysis results of SDS-PAGE showed that the purity of obtained Kringle 5 was about 98%.After desalting with size-exclusion chromatography,it was found that it inhibited the blood vessel growth of chick embryo chorioallantoic membrane effectively.This simple,effective method may be applied in the future in mass production of Kringle 5 and lay a solid base for detailed evaluation of bioactivities of Kringle 5.