Construction And Identification Of Eukaryotic Coexpression Vector PIRES-hVEGF₁₂₁/hBMP-4

Objective To construct the eukaryotic coexpression vector encoding human vascular endothelial growth factor (VEGF121) and human bone morphogenesia protein-4 (hBMP-4), and detect the expression of the plasmid after COS-7 cell being transformed into.Methods Firstly, VEGF121 was directly cloned into one of the multiple cloning sites of the eukaryotic expression plasmid-pIRES to construct pIRES-hVEGF121 . Secondly,hBMP-4 was directly cloned into pShuttle CMV to construct pShuttle CMV-hBMP-4, a new reconstructed shuttle plasmid. Carried with the instrument of pShuttle CMV-hBMP-4,hBMP-4 was cloned into pIRES-hVEGF121 to construct pIRES-hVEGF121/hBMP-4, which laterly were confirmed by restriction enzymolysis and then transformed into COS-7. Thirdly, the transcription of the RNA extracted from COS-7 were detected by RT-PCR and the expression of proteins extracted from COS-7 were detected by Western blot assay.Results The eukaryotic expression plasmid-pIRES-hVEGF121 could be digested by enzyme, which can certificate its right construction; the shuttle plasmid-pShuttle CMV-hBMP-4 could be digested by enzyme, which can certificate its right construction; the eukaryotic expression plasmid-pIRES-hVEGF121/hBMP-4 could be digested by enzyme, which can certificate its right construction. The transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay.Conclusion The construction of pIRES-hVEGF121/hBMP-4 was acheved; the transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay; the proteins expression of hBMP-4 and hVEGF121 genes could set a sound ground for the further researches.