CARP基因重组腺病毒载体的构建

Construction of Recombinant Adenovirus Vector of CARP

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 2 93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 2 93细胞 3天后可见绿色荧光 ,回收病毒可以重复感染 2 93细胞 。

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. coll. 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. Resuits： 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.