Objective To be expressed human vascular endothelial growth factor (VEGF) recombinant protein in Escherichia Coli in high level.
Methods VEGF was amplified from human fetal brain cDNA library, the amplified fragment was inserted into M13mP18 and confirmed to be VEGF165cDNA by restriction mapping and DNA sequencing, then it was combined with an expression vector PRL621. This recombinant plasmid overexpressed a 20kd recombinant protein in E.Coli(TG1), the protein was isolated and purifed from E.Coli, and initially renatured.
Results The overexpressed recombinant protein was 35% of the total cell protein, the sequence of its first 15-N terminal amino acid was identrical to that of the human natural VEGF protein, Chorioallantoic membrane(CAM) assay showed that the rhVEGF promated new capillary vessels formation.
Conclusion The genetic engineering Escherichia Coli can express human vascular endothelial growth factor in high level.
Citation：
HE Yanzheng,LIU Jianping,XIE Yi,et al.. CLONING cDNA OF HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR AND HIGH LEVEL EXPRESSION IN ESCHERICHIA COLI. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2001, 8(2): 72-74. doi:
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| 1. |
Anne A, Knud SC. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia [J]. Circulation, 1999; 99(17)∶2967.
|
| 2. |
何延政，张培华，刘建平等.重组血管内皮生长因子在大肠杆菌的高效表达 [J].中华实验外科杂志，1997； 14(5)∶263.
|
| 3. |
何延政，张培华.血管内皮细胞和血管病的基因治疗 [J].中华实验外科杂志，1995； 12(增刊)∶119.
|
| 4. |
何延政，张培华.腺病毒介导的体内基因转移 [J].中华实验外科杂志，1995； 12(增刊)∶121.
|
| 5. |
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual [M]. Second edition, New York:Cold Spring Laboratory Press, 1989∶16-688.
|
| 6. |
Leung DW, Cachianes G, Kuang W J, et al. Vascular endothelial growth factor is a secreted angiogenesis mitogen [J]. Science, 1989; 246(4935)∶1306.
|
| 7. |
Shen, BO. Homologous upregulation of KDR/Flk1 receptor expression by vascular endothelial growth factor in vitro [J]. J Biol Chem, 1998; 273(6)∶29979.
|
| 8. |
Mohanraj D. Expression of biologicaly active human vascular endothelial growth factor in yeast [J]. Growth Factor, 1995; 12(1)∶17.
|
| 9. |
Perets D, Goren HG, Safram M. Glyosylation of vascular endothelial growth factor is required for its mitogenic activity [J]. Biochem Biophys Res Commun, 1992; 182(3)∶1340.
|
| 10. |
Yeo TK. Glyosylation is essential for efficient secretion but not for VEGF [J]. Biochem Biophys Res Commun, 1992; 179(3)∶1568.
|
- 1. Anne A, Knud SC. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia [J]. Circulation, 1999; 99(17)∶2967.
- 2. 何延政，张培华，刘建平等.重组血管内皮生长因子在大肠杆菌的高效表达 [J].中华实验外科杂志，1997； 14(5)∶263.
- 3. 何延政，张培华.血管内皮细胞和血管病的基因治疗 [J].中华实验外科杂志，1995； 12(增刊)∶119.
- 4. 何延政，张培华.腺病毒介导的体内基因转移 [J].中华实验外科杂志，1995； 12(增刊)∶121.
- 5. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual [M]. Second edition, New York:Cold Spring Laboratory Press, 1989∶16-688.
- 6. Leung DW, Cachianes G, Kuang W J, et al. Vascular endothelial growth factor is a secreted angiogenesis mitogen [J]. Science, 1989; 246(4935)∶1306.
- 7. Shen, BO. Homologous upregulation of KDR/Flk1 receptor expression by vascular endothelial growth factor in vitro [J]. J Biol Chem, 1998; 273(6)∶29979.
- 8. Mohanraj D. Expression of biologicaly active human vascular endothelial growth factor in yeast [J]. Growth Factor, 1995; 12(1)∶17.
- 9. Perets D, Goren HG, Safram M. Glyosylation of vascular endothelial growth factor is required for its mitogenic activity [J]. Biochem Biophys Res Commun, 1992; 182(3)∶1340.
- 10. Yeo TK. Glyosylation is essential for efficient secretion but not for VEGF [J]. Biochem Biophys Res Commun, 1992; 179(3)∶1568.
