Construction of Cloned Recombinant Adenovirus Genome by Homologous Recombination in Escherichia coli

To efficiently construct cloned human type 5 recombinant adenovirus bearing exogenous rabies glycoprotein gene by employing homologous recombination in Escherichia coli .The rabies glycoprotein gene(GP)was first subcloned into shuttle vector pAd Track CMV in which an enhanced green fluorescent protein gene(EGFP)expression cassette had been incorporated in advance.The resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 cells by electroporation with adenoviral backbone plasmid pAdEasy 1.To directly confirm recombination,conventional DNA sequencing was performed with the double strand plasmid DNA as template and oligonucleotide synthesized as GP specific primer.After the cloned recombinant adenovirus plasmid DNA was obtained,it was digested with Pac I for releasing ITR and transfected into package 293 cell to get recombinant virus.Genomic homogeneous recombinant adenovirus was tested by electron microscope for morphology and fluorescent microscope for detection of the expressed EGFP.As a result,DNA sequencing indicated that GP gene was successfully integrated into the target region of the adenoviral genome.Homogenous recombinant virus could be easily obtained by transfecting 293 cell with this infectious DNA.The resultant recombinant adenonovirus could reach 1×10 8 pfu/ml in 293 cells and showed typical adenovirus morphological characteristics with a diameter about 70 nm when observed by electron microscope.They also expressed tremendous EGFP in infected cells.These experiments suggest that construction of cloned recombinant adenovirus genome by homologous recombination in Escherichia coli is a very efficient method to construct homogeneous recombinant adenovirus.