IL-7和EGFP双基因共表达重组腺病毒载体的构建

目的：构建白细胞介素7（IL-7）和增强型绿色荧光蛋白（EGFP）共表达的重组腺病毒载体,为进一步感染干细胞奠定基础。方法：将目的基因IL-7克隆到含有报告基因EGFP的穿梭质粒中,然后再将构建的重组穿梭质粒转移至pAdxsi载体中,构建重组腺病毒载体质粒,继而在H293细胞中扩增,纯化后测定病毒滴度。结果：pShuttle-EGFP-mIL7重组穿梭质粒经酶切鉴定得到0.5kb和5.1kb 2条带;pAdxsi-EGFP-mIL7重组腺病毒载体质粒经酶切鉴定得到14K、11.8K、3.1kb、2.66kb、2.47K、1.45K、0.6K 7条带;TCID50法测定纯化后的病毒滴度为2×10^10pfu/ml。结论：pAdxsi-EGFP-mIL7重组腺病毒载体构建成功。

Construction of recombinant adenoviral vector co-expressing interleukin-7 and enhanced green fluorescent protein

Objective：To construct the adenoviral vector co-expressing interleukin-7（IL-7） and enhanced green fluorescent protein（EGFP）,to lay a experimental foundation for further study on the infection into stem cell.Methods： The target gene IL-7 was cloned into the shuttle plasmid expressed the report gene EGFP.Then the recombinant shuttle plasmid was transformed into Dh5a bacteria to recombine with backbone vector pAdxsi.Next,the plasmid pAd-EGFP-mIL7 was amplified in H293 cells and purifired,then the viral titer was determined.Results： The recombinanted shuttle plasmid pShuttle-EGFP-mIL7 digested with restriction endonucleases was confirmed by two products which length were respectively about 0.5kb and 5.1kb;the recombinanted plasmid pAdxsi-EGFP-mIL7 digested with restriction endonucleases was confirmed by seven products which length were respectively about 14K,11.8K,3.1kb,2.66kb,2.47K,1.45K and 0.6K;recombinant adenoviral amplifired with titer of 2×10^10pfu/ml.Conclusion： The recombinant adenoviral vector pAdxsi-EGFP-mIL7 was successfully constructed.