人SNAP25基因重组腺病毒的构建与表达

Construction and Expression of SNAP25 Gene Mediated by Recombinant Adenovirus

利用RT-PCR法,从人胰腺组织扩增出SNAP25基因,定向克隆至pENTR-TOPO载体获得重组质粒pENTR/D-TOPO-SNAP25。经PCR及测序验证其阅读框正确。再与腺病毒载体pAD/CMV/V5-DEST发生LR重组反应,SNAP25取代pAD/CMV/V5-DESTTM中的ccdB-CmR基因,获得重组腺病毒载体pAD/CMV/V5-DEST-SNAP25(pAD-SNAP25)。重组腺病毒载体经PacI酶切,脂质体法转染HEK-293A细胞.以鼠抗人SNAP25单克隆抗体为一抗,做荧光及Westernblot检测,结果表明重组腺病毒pAD-SNAP25在HEK-293A细胞中包装成功。重组腺病毒pAD-SNAP25感染大鼠胰岛,结果表明在高糖浓度下,外源性SNAP25蛋白的表达促进了大鼠胰岛素分泌。

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- DEST-SNAP25, which were detected with mouse anti-human monoclonal SNAP25 antibody in transfected HEK- 293A cells. The results indicate that recombinant adenovirus Rat islet β-cells infected with adenovirus pAD-SNAP25, level pAD-SNAP25 was packaged in HEK-293A cells. of insulin secretion were enhanced by expression of exogenous SNAP-25 at high glucose concentration.