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Construction and identification of recombinant adenovirus vector carrying rat GLUT3 gene
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 11
- DOI:
- 10.3760/cma.j.issn.1671-8925.2006.11.003
- Key Word:
- 葡萄糖转运体3;基因克隆;重组腺病毒
Abstract： 目的 构建携带大鼠葡萄糖转运体3(GLUT3)基因的复制缺陷型重组腺病毒载体,为研究GLUT3的生物学功能提供工具.方法 采用逆转录-聚合酶链反应(RT-PCR)的方法获取大鼠GLUT3基因全长cDNA片段,将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-GLUT3,经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α,构建重组腺病毒质粒pAd-GLUT3,酶切线性化重组腺病毒质粒后转染293细胞包装成重组病毒颗粒,重组腺病毒在293细胞中反复扩增数代后,分别用聚合酶链反应(PCR)及免疫印记法(western blot)的方法从基因和蛋白表达水平鉴定重组的腺病毒.结果 经DNA测序和PCR分析显示GLUT3 cDNA序列正确.成功筛选出重组腺病毒质粒pAd-GLUT3后在HEK293细胞中成功包装出重组病毒,包装后冻融细胞行PCR及western blot检测表明重组腺病毒包装成功.结论 本研究成功构建了携带大鼠GLUT3基因的复制缺陷型重组腺病毒载体.
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