High efficient generation of recombinant adenovirus by bacterial intermolecular homologous recombination

Objective:To construct recombinant adenovirus plasmid using homologous recombination in E.coli strain BJ 5183 by two-step transformation procedure and to prepare recombinant adenovirus particles.Methods:Adenovirus backbone plasmid was transformed into competent BJ 5183 and the competent BJ 5183 transformed with pAdEasy-1 was prepared.The linearized pAdtrack-CMV with P meI digestion and CIAP dephosphorylation was transformed into the competent BJ 5183 transformed with pAdEasy-1.The identified recombinant adenovirus plasmid pAd Eazy-CMV was digested with Pac Ⅰ and transfected into HEK293 cells to package recombinant adenovirus particles RAd-CMV,and the recombinant adenovirus particles was identified by transmission electron microscopy and purified using cesium choride density centrifugation,then viral tite was checked by GFP.Finally,HeLa cells were infected by the recombinant adenovirus.Results:Recombinant adenovirus vector pAd-CMV was constructed by homologous recombination in E.coli,and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-CMV.The high expression of green fluorescence protein expression in HEK293 and HeLa cell lines were found under fluorescent microscope.Electron microscopy test indicated that a recombinant adenovirus RAd-CMV was successfully constructed,and the titer of the recombinant adenovirus reached 2.0×1010pfu/ml.Conclusion:Homologous recombination between adenoviral backbone plasmid and shuttle plasmid containing GFP gene within bacteria should speed up the process of generating recombinant adenoviruses and avoid the plaque purification,which lays the foundation for further researches of construction of recombinant adenovirus expressing therapeutic gene of interest.