【摘要】 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AWP1cDNA插入高效表达RFP的载体pDsRed1-N1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PmeI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PacI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。更多还原

【Abstract】 Objective To construct AWP1-RFP fused gene recombinant adenovi rus so as to further investigate the biological functions of human AWP1 (associa ted with protein kinase C related kinase 1). Methods AWP1 cDNA was firstly subcloned into the multiply clone sites (MCS) of pDsRed1-N1 plasmid expressing red fluorescence protein (RFP). Then AWP1-RFP fused gene was secondly subcloned into the shuttle vectors pAdTrack-CMV and pAdShuttle-CMV. After identification with restrictional enzymes, the recombinant plasmids were linearized by digestio n with restriction endonuclease PmeⅠ,and subsequently cotransformed into E.coli BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1 to make homologous r ecombination. The homologous recombinant plasmids linearized by PacⅠ were packa ged in 293 cells transfected with lipofectAMINE. The identifications of recombi nant adenovirus were performed by the methods of DNA sequencing and PCR assays o f recombinant vectors and packaged adenoviruses granules and AWP1-RFP fusion pro tein expression in 293 cells. Results DNA sequencing and PCR assays showed that the sequence of the AWP1 cDNA was correct. AWP1-RFP fused protein was expr essed high-efficiently in 293 cells infected by homologous recombinant viruses. Conclusion AWP1-RFP fusion gene recombinant adenoviruses were successfully c onstructed and packaged.更多还原