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／V5一DEST 发生LR重组反应，SNAP25取代pAD／CMV／V5一DESTTM中的ccdB-CmR基因，获得重组腺病毒载体pAD／CMV／V5一DEST-SNAP25(pAD- SNAP25)。重组腺病毒载体经Pac I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Western Blot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.
庄国庆,王有利,马军丽,刘芬,王嵬,何燕. 人SNAP25基因重组腺病毒的构建与表达[J]. 中国生物工程杂志, 2008, 28(10): 18-22.
. Construction and Expression of SNAP25 Gene mediated by Recombinant Adenovirns. China Biotechnology, 2008, 28(10): 18-22.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I10/18
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