Comparison of the Co-expression Level of Ang-1/VEGF165 Mediated by IRES and PolyA-promoter

Objective:Construct recombinant adenoviral vector co-expressing angiogenin-1(Ang-1)and vascular endothelial growth factor 165(VEGF165)mediated by IRES and polyA-promoter,then compare the expression of Ang-1/VEGF165 mediated by IRES and polyA-promoter as well as in the downstream and upstream of polyA-promoter/IRES likewise their vascular inducibility,to provid experimental evidence for constructing superior recombinant vector co-expressing double gene or multiple gene.Methods:The VEGF165 and Ang-1 fragments were amplified by PCR using pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmids as templates.The VEGF165 and Ang-1 fragments were subcloned into pAdTrack-CMV-IRES and pAdTrack-CMV-PolyA-promoter transfer vector,and then identified by PCR,double endonuclease digestion,and DNA sequencing.The pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV-VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1 gene recombinated transfer vectors were cotransformed into the bacteria BJ5183 competent cells with pAdEasy-1 backbone vector for homologous recombination,then they were linearized with PacI digestion and transfected into the human embryonic kidney 293(293A)cells,leading to formation of the recombinant adenoviruses Ad-Ang-1-polyA-promoter-VEGF165、 Ad-VEGF165-polyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1.The transcription of VEGF165 and Ang-1 were identified by RT-PCR,and the expression of VEGF165 and Ang-1 in different adenoviral vector were then identified by enzyme-labeled immunosorbent assay(ELISA),then compared the expression level of Ang-1 and VEGF165 gene in IRES and polyA-promoter mediated vector as well as in the downstream and upstream of the same adenoviral vector.Injected Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165 to the rabbit corneal limbal and detected the new vessels areae,then compared their vascular inducibility.Results:The sequencing of Ang-1 and VEGF165 were correct,three kinds of recombinant adenoviral vector were all successfully constructed and obtained,and their potency were up to 2~5×1010pfu/ml,the transcription of VEGF165 and Ang-1 were also significant by RT-PCR.Moreover VEGF165 and Ang-1 gene expressing in the WI-38 cells was confirmed by ELISA,also found that the IRES-mediated Ang-1 and VEGF165 gene,either upstream or downstream,its expression level was lower than the same location in polyA-promoter-mediated genes,degraded about 60%~70%,moreover either the IRES vector or polyA-promoter vector,the expression of downstream genes volume was significantly lower than the upstream gene volume,degraded about 30%~40%.At the same time the animal experiment of vasiformation in rabbit cornea suggestted that the vascular inducibility of Ad-VEGF165-PolyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1 were better,and the former was stronger than the latter.Conclusion:In the adenoviral expression vector,Ang-1 and VEGF165 gene could both successfully expressed in cell,and both had vascular inducibility.The expression level and vascular inducibility of polyA-promoter-mediated double gene was higher and strongger than the IRES-mediated double gene,at the same time the expression level and vascular inducibility of the gene volume in the downstream of polyA-promoter/IRES were lower than the genes volume in the upstream.