中国生物工程杂志

To construct and express human SNAP25 gene mediated by recombinant adenovirus vectors．human SNAP25 gene was coloned by RT-PCR and directly cloned into vector Pentr-TOPO to fulfill TOPO-SNAP25 plasmid.The recombinant plasmid TOPO-SNAP25 was identified and confirmed by PCR and sequencing.SNAP25 gene was cloned into the pAD／CMV／V5-DESTTM gateway vector by LR recombination reaction with pAD／CMV／V5-DESTTM gateway vectors and TOPO-SNAP25 plasmid. The ccdB-CmR gene in pAD／CMV／V5-DESTTM was replaced by SNAP25 gene.The recombination vector was digested by Pac I enzyme and transfected into HEK-293A cells by Lipofectamine 2000 to obtain recombinant adenovirus vectors pAD／CMV／V5一DEST-SNAP25, which were detected with mouse anti-human monoclonal SNAP25 antibody in transfected HEK-293A cells. The results indicate that recombinant adenovirus pAD-SNAP25 was packaged in HEK-293A cells. Rat islet β-cells infected with adenovirus pAD-SNAP25, level of insulin secretion were enhanced by expression of exogenous SNAP-25 at high glucose concentration.