Construction Of AAV Co-expression System For Human Angiopoietin1 And VEGF Gene

Objective In clinical practice , ischemic disease is difficult to cure. Utilizing transgenic technology to stimulate healthy and effective neovascularization in ischemic tissue is the hot spot on the study of ischemic disease. Recent study showed that vascular endothelial growth factor (VEGF) and angiopoietin 1 gene plays important role in the early and late stage of neovascularization separately. Adeno-associated virus is a hopeful gene delivery system in gene transferring. A recombinant plasmid for co-expression of human angiopoietin 1 and VEGF 165 gene in AAV gene delivery system was constructed in this topic to provide a foundation for gene therapy of severe ischemic disease. Methods Based on the original sequence provided by NCBI Gene Bank, primer Pangl and Pang2 were designed and the cDNA Library of bone marrow was acted as pattern , human angiopoietin 1 was obtained by polymerase chain reaction (PCR). Based on the original sequence provided by NCBI Gene Bank, primer Pvegfl and Pvegf2 were designed and the plasmid phVEGF was acted as pattern ,human VEGF 165 was obtained by PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hindlll, the downstream of angiopoietin 1 contained restriction enzyme site BamH I. The upstream of VEGF 165 contained restriction enzyme site Bgl II, the downstream of VEGF 165 contained restriction enzyme site BamH I. The multiple cloning sites(MCS) in plasmid pAAV-MCS including BamH I, Bgl II, Hindlll, Not I, Xho I, Xba I, Sal I, BspH I, Ksp I , while the plasmid pZero++ contained almost all common cleavage sites. Using this specialty and using pZero++ as a shuttle plasmid, angiopoietin 1 and VEGF 165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF 165 were identical with NCBI Gene Bank. The recombinant plasmid pAAV VEGF, pAAV Angiopoietin 1 and pAAV Angiopoietin 1-VEGF were identified by DNA sequencing and digestion, which proved to be consistent with our hypothesis. In recombinant plasmid pAAV Angiopoietin 1-VEGF, angiopoietinl and VEGF possessed a CMV promoter and polyA terminator system respectively, which assured co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy of severe ischemicdisease.