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

摘要

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 heal -ing .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 DsbA which had 6*His tag in N terminal .Inter-estingly, the addition of 5%(v/v) ethanol to the culture medium resulted in the production of large amounts of soluble DsbA-VEGF165 fusion protein .The VEGF165 fusion protein was subjected to a Ni-NTA affinity chromatog-raphy followed by enterokinase digestion .The rhVEGF165 was finally purified by Heparin-Sepharose affinity chro-matography .The biological activity of rhVEGF 165 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 .%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 （DsbA）的E．coli表达系统实现了 DsbA-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 kDa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／mL。研究实现了DsbA-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。