血管内皮生长因子在原核细胞中的高效表达、纯化与活性研究(英文)

Prokaryotic expression, purification and activity assay of recombinant vascular endothelial growth factor

目的用基因工程的方法在大肠杆菌中诱导表达人血管内皮生长因子(VEGF165),分离纯化并检测其免疫学以及生物学活性,为后期构建携带VEGF165蛋白的预成血管化组织工程骨提供充足的蛋白来源。方法利用PCR亚克隆的方法获得目的基因片段,构建表达质粒pQE30-VEGF165,在大肠杆菌JM109中用IPTG进行诱导表达并使用Ni2+-NTA进行蛋白纯化。酶联免疫吸附(ELISA)和Westernblot技术检测纯化的VEGF165蛋白免疫学活性,鸡胚尿囊绒毛膜实验和matrigel血管形成实验检测其生物学活性。结果重组表达质粒pQE30-VEGF165在大肠杆菌中成功的表达了相对分子质量为23000的蛋白,该蛋白以不溶性包涵体的形式存在,占菌体总蛋白的30%左右。经过分离和Ni柱纯化获得了VEGF165蛋白,浓度约为0.2mg/ml,ELISA和Westernblot实验显示其具有良好的免疫学活性,鸡胚尿囊绒毛膜实验和matrigel血管形成实验显示所表达的蛋白具有良好的生物学活性。结论本实验在原核细胞中稳定、高效的表达了具有活性的VEGF165蛋白,为后期预购血管化组织工程骨的构建提供了充足的蛋白来源。

Objective To express human vascular endothelial growth factor （hVEGF165） in E.coli JM109 in the form of fusion protein by genetic engineering and test the biological activity and immunological competence of the expressed protein. Methods hVEGF165 gene was subcloned by PCR and inserted into pQE30 plasmid, hVEGF165 fusion protein was expressed in E.coli JM109 and purified by Ni2^＋-NTA. The immunological competence of the expressed protein was tested by means Western blotting and enzyme-linked immunosorbent assay （ELISA）, and its biological activity was assayed by chicken chorioallantoic membrane （CAM） and Matrigel angiogenesis assay. Results The recombinant hVEGF165 fusion protein was successfully expressed in E.coli JM109 and its expression naccounted for 30% of the total cellular protein. The purified protein presented a single band of 23 kD in SDS-PAGE. Western blotting, ELISA, CAM and matrigel angiogenesis assay showed excellent immunologic competence and biological activity of the recombinant protein. Conclusion Recombinant hVEGF165 protein with excellent biological activity has been successfully expressed in E.coli JMI09, which may facilitate future study in construction of prefabricated tissue-engineered bone graft.