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Rapid construction of AWP1-RFP fused gene recombinant adenovirus vector and its identification
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 5
- DOI:
- 10.3760/cma.j.issn.1671-8925.2005.05.002
- Key Word:
- AWP1;红色荧光蛋白;重组腺病毒;绿色荧光蛋白
Abstract： 目的构建人的AWP1与红色荧光蛋白(RFP)融合基因的重组腺病毒载体,为研究AWP1的生物功能提供工具. 方法将克隆的AWP1 cDNA插入高效表达RFP的载体pDsRed1N1的多克隆位点,与RFP序列形成AWP1-RFP融合基因,将该融合基因亚克隆到穿梭载体pAdTrack-CMV和pAdShuttle-CMV中;经酶切鉴定正确后Pme Ⅰ线形化,与骨架质粒pAdEasy-1共转染大肠杆菌BJ5183以同源重组;用lipofectAMINE将Pac Ⅰ线形化的同源重组腺病毒载体转染293细胞以包装腺病毒;通过同源重组病毒载体和病毒颗粒基因组的DNA测序及PCR扩增和在293细胞中的表达鉴定重组腺病毒.结果同源重组载体和病毒颗粒的DNA测序和PCR分析显示AWP1cDNA序列正确,感染293细胞获得了AWP1-RFP融合蛋白的高效表达.结论成功构建和包装了人AWP1-RFP融合基因重组腺病毒.
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