重组人血管内皮生长因子165在大肠杆菌中的可溶性表达、纯化与体外活性评价  

Prokaryotic Soluble Expression and Purification of Functional Human Vascular Endothelial Growth Factor 165

作　　者：周晓雷[1] 朱重悦[1] 张世光[1] 李海潮[1] 张竞 王岩勃 邹卫[1] 

机构地区：[1]河北科技大学 [2]石家庄沃泰生物科技有限公司,石家庄050018

出　　处：《科学技术与工程》2016年第1期42-46,共5页Science Technology and Engineering

基　　金：河北省科技计划项目(15272401D);河北科技大学五大平台开放基金课题(SW05)资助

摘　　要：人血管内皮生长因子165(VEGF165)可有效促进血管新生和增加血管通透性,在伤口愈合方面有重要医疗价值。建立获取高纯度、高活性的优质重组VEGF165蛋白的方法具有重要意义。研究利用带有6组氨酸标签的二硫键形成蛋白A(Dsb A)的E.coli表达系统实现了Dsb A-VEGF165融合蛋白的可溶性表达;诱导过程中添加5%(v/v)的乙醇可显著提高工程菌中可溶性融合蛋白表达水平。融合蛋白通过Ni亲和层析粗纯,并经牛肠激酶酶切去除标签蛋白。随后利用肝素亲和层析精纯获得重组人VEGF165蛋白。非还原及还原SDS-PAGE电泳检测到分子量为约40 k Da的同源二聚体蛋白,促HUVEC细胞增殖实验显示重组蛋白具有较优的活性,EC50为13 ng/m L。研究实现了Dsb A-VEGF165的在E.coli中可溶性表达,建立了经济、高效的纯化方法,获得了高质量、高活性的重组人VEGF165蛋白。Vascular endothelial growth factor（ VEGF165） is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for acceleration wound healing. An production method that provides high yield as well as high purity,quality,and potency is needed. Soluble VEGF165 protein was expressed in Escherichia coli by fused with Dsb A which had 6＊ His tag in N terminal. Interestingly,the addition of 5%（ v / v） ethanol to the culture medium resulted in the production of large amounts of soluble Dsb A-VEGF165 fusion protein. The VEGF165 fusion protein was subjected to a Ni-NTA affinity chromatography followed by enterokinase digestion. The rh VEGF165 was finally purified by Heparin-Sepharose affinity chromatography. The biological activity of rh VEGF165 is comparable with VEGF from eukaryotic source according to human umbilical vein endothelial cells（ HUVEC） proliferation assay. The present procedures provide a fast and easy way to produce this therapeutic protein.

关 键 词：人血管内皮生长因子165 融合蛋白 原核表达 蛋白纯化 

分 类 号：Q786[生物学—分子生物学] Q812