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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。
Objective To construct a recombinant adenovirus vector containing human fusion gene AWP1 and red fluorescent protein (RFP) to provide a tool for studying the biological function of AWP1. Methods The cloned AWP1 cDNA was inserted into the multiple cloning site of pDsRed1-N1 highly expressing RFP to form AWP1-RFP fusion gene with RFP sequence. The fusion gene was subcloned into shuttle vector pAdTrack-CMV and pAdShuttle-CMV. After identification of the correct PmeI linearization, co-transfection with the backbone plasmid pAdEasy-1 E. coli BJ5183 for homologous recombination; lipofectAMINE PacI linearized homologous recombinant adenovirus vector transfected 293 cells to package adenovirus; by homologous DNA Sequencing and PCR Amplification of Recombinant Viral Vector and Viral Particle Genomes and Expression in 293 Cells Identification of recombinant adenoviruses. Results DNA sequencing and PCR analysis of the homologous recombination vector and virus particles showed that the AWP1 cDNA sequence was correct. The 293 cells were highly expressed in AWP1-RFP fusion protein. Conclusion The human AWP1-RFP fusion gene recombinant adenovirus was successfully constructed and packaged.