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Construction and Expression of SNAP25 Gene mediated by Recombinant Adenovirns |
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Abstract 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.
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Received: 19 May 2008
Published: 25 October 2008
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