Construction of Adenovirus Vector with Human Interleukin 10 and Its Expression in Vascular Smooth Muscle Cells

Objective To construct recombinant adenovirus vector co-expressing human interleukin(hIL)-10 and green fluorescent protein(GFP) for study of the expression of genes of interest in vascular smooth muscle cells(VSMCs).Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction(PCR),and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1.Kanamycin resistance screeninged for recombinant plasmids,which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert's sequence correct by NotⅠ and XholⅠdigestion,sequencing and basic local alignment search tool(BLAST).Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells.Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ.PCR was used to determine target gene;The titer of the recombinant adenovirus was measured.VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope.hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay(ELISA).Results Recombinant shuttle plasmids contained interest gene.Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ.PCR indicated that the recombinant adenovirus contained interest gene.The titer of recombinant adenovirus was 3×1010 efu/ml.Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells.Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs,this lays the foundation for the gene therapy of vascular intimal hyperplasia.