Journal of Southern Medical University

Journal of Southern Medical University ›› 2006, Vol. 26 ›› Issue (09): 1263-1268.

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

YANG Pei1, WANG Kun-zheng1, SHI Zhi-bin1, DANG Xiao-qian1, YU Peng-bo2, WANG Chun-sheng1, GONG Fu-liang3 1Department of Orthopedics, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an 710004, China; 2Institute of Virus Research, Center for Disease Control and Prevention of Shaanxi Province, Xi’an 710054, China; 3Department of Orthopedics, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China   

  1. 西安交通大学医学院第二附属医院骨科； 陕西省疾病预防控制中心病毒室； 大连医科大学附属第一医院骨科 陕西西安710004； 陕西西安710004； 陕西西安710054； 辽宁大连116011；
  • Online:2006-09-20 Published:2006-09-20

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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 hVEGF165 protein 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. 

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