CARP基因重组腺病毒载体的构建

目的：利用Adeasy-1系统，构建并鉴定CARP基因重组腺病毒载体。方法：PCR扩增含有CARP全长cDNA的片段，经测序验证无误后，亚克隆至pAdTrack-CMV穿梭质粒，再与pAdEasy．1质粒在大肠杆菌BJ5 183中进行同源重组产生腺病毒载体质粒。经过抗性筛选以及酶切鉴定得到阳性的重组质粒，再在293细胞中进行包装扩增，利用Adeasy系统上的绿色荧光蛋白标签鉴定病毒表达。结果：测序证实PCR产物为CARP全长cDNA；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；转染293细胞3天后可见绿色荧光，回收病毒可以重复感染293细胞，证明病毒包装成功。

Construction of Recombinant Adenovirus Vector of CARP

Objective:To develop a gene therapy vector of CARP using recombinant adenovirus.Methods:PCR was performed to get full-length cDNA of CARP gene.The gene was then subcloned into the pAdTrack-CMV shuttle vector.The resultant plasmid (pAdTrack-CMV-CARP) was cotransduced into E.coli.BJ5183 cells with pAdEasy-1 plasmid to undergo homologous recombination.The linearized recombinant plasmid (pAd-CARP) was transfected into 293 cells.The recombinant adenovirus was detected by examining the expression of the GFP.Results:By sequencing,it was confirmed that the PCR product was a full-length cDNA of CARP.Restriction endonuclease analysis confirmed the successful cloning of the gene into the pAdTrack-CMV and the recombinants (pAd-CARP) were selected for kanamycin resistance.Presence of the recombinant adenoviruses was confirmed by GFP expression.