Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene.
BACKGROUND
Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165(VEGF165) gene in adeno-associated virus (AAV) gene delivery system.


METHODS
Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site HindIII, and the downstream of angiopoietin 1 contained restriction enzyme site BamHI. The upstream of VEGF165 contained restriction enzyme site BglII, and the downstream of VEGF165 contained restriction enzyme site BamHI. Using the multiple cloning sites (MCS) in plasmid pZero++ such as BamHI, BglII, HindIII, NotI, XhoI, XbaI, SalI, BspHI, KspI and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.


RESULTS
DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion, which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.


CONCLUSIONS
Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.
Chen
De-Jie
DJ
Department of Vascular Surgery, Zhongnan Hospital, Wuhan University, Wuhan 430071, China.
Tan
Zui
Z
Xie
You-Li
YL
Liu
Fang
F
D042683:Angiopoietin-1 / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D042461:Vascular Endothelial Growth Factor A