核糖体蛋白L8重组腺病毒载体的构建及鉴定

摘要

目的:构建鼠核糖体蛋白L8(RPL8)基因的重组腺病毒载体,为转染小鼠树突状细胞(DC)为制备DC疫苗奠定基础.方法:用RT-PCR克隆目的基因RPL8,与载体连接、酶切、插入、并转移到pAdxsi腺病毒骨架载体上,包装扩增成腺病毒颗粒.用重组腺病毒及空病毒Ad-EGFP(作为阴性对照)转染小鼠DC细胞,荧光显微镜下观察细胞绿色荧光蛋白的表达.结果:经酶切及测序鉴定,成功构建了携带RPL8基因的重组腺病毒载体,并制备出高滴度(1.6 × 1010 PFU/mL)的重组腺病毒.结论:成功构建了携带RPL8的重组腺病毒,为进一步研究RPL8基因修饰DC疫苗奠定基础.%Objective To construct the recombinant adenovirus vector encoding mouse RPL8 gene for transfecting dendritic cells (DC). Methods RPL8 gene was cloned by RT-PCR, and after being processed, the gene was transferred into pAdxsi adenovirus vector and was packed into adenovirus gramule. Then the recombinant adenovirus and Ad-EGFP were transfected in mice DC, the expression of the green fluorescencent protein and RPL8 were detected by fluorescencent microscope. Results As confirmed by restriction digestion analysis, an expectant was observed in proper recombinant adenovirus vector encoding RPL8 gene. The viral titer checked by GFP was about 1.6×1010 PFU/mL. Conclusions The recombinant adenovirus vector encoding RPL8 gene has been established successfully.