携带内皮抑素基因的重组腺病毒的构建

Construction of a recombinant adenovirus carrying endostatin gene

目的利用AdEasy-1系统,构建并鉴定人内皮抑素重组腺病毒,为后续的实验奠定基础。方法以PshuttleEndostatin质粒为模版PCR扩增内皮抑素基因片断,克隆至穿梭质粒pAdTrack-CMV中,再与骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组,在AAV293细胞中包装并扩增。结果重组腺病毒经测序、酶切鉴定正确,病毒滴度为2.06×1010pfu/ml。结论人内皮抑素重组腺病毒pAd-Endo构建成功。

Objective To construct a recombinant adenovirus carrying human endostatin gene with AdEasy system.Methods Endostatin gene fragment was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector.The resultant plasimid was cotransduced into E.coli BJ 5183 cells with pAdEasy-1 plasmid for homologous recombination.The linearized recombinant plasmid was subsequently transfected into AAV 293 cells,and the recombinant adenovirus was detected by GFP,PCR and restriction analysis.Results and Conclusion The positive clones of the recombinants were verified by restriction analysis and the titer of the virus reached 2.06×10^10 pfu/ml,suggesting successful construction of recombinant adenovirus pAd-Endo.