Prokaryotic expression, purification and activity assay of recombinant vascular endothelial growth factor
10.3321/j.issn:1673-4254.2006.09.001
- VernacularTitle: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
- Author:
Pei YANG
1
;
Kun-Zheng WANG
;
Zhi-Bin SHI
;
Xiao-Qian DANG
;
Peng-Bo YU
;
Chun-Sheng WANG
;
Fu-Liang GONG
Author Information
1. 西安交通大学附属第二医院
- Keywords:
vascular endothelial growth factor;
protein expression;
purification
- From:
Journal of Southern Medical University
2006;26(9):1263-1268
- CountryChina
- Language:Chinese
-
Abstract:
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 of 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 accounted 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 hVEGF165protein with excellent biological activity has been successfully expressed in E. coli JM109, which may facilitate future study in construction of prefabricated tissue-engineered bone graft.
