Construction and identification of recombinant adenovirus with GFP labeled protein IX

Objective To study the role of protein IX(pIX) in the interaction between adenovirus and host cell dynamically, we attempt to construct a recombinant human adenovirus with the capsid protein pIX being labelled with Green Fluorescent Protein(GFP). Methods GFP gene was inserted into the downstream of pIX genein the shuttle plasmid to form pShuttle-IXGFP; and adenoviral plasmid pAd5-IXGFP was generated by homologous recombination of pShuttle-IXGFP with the backbone plasmid (pAdeasy-1) in E. coli BJ5183 strain. Recombinant adenovirus (Ad5-IXGFP) was rescued by transfecting 293 cells with the linearized pAd5-IXGFP. Results After 4 rounds of propagation in 293 cells, viruses were purified with CsCl ultracentrifugation method. Ad5-IXGFP in 1.0 ml with a particle titer of 6.9×1011 vp/ml was finally obtained. PCR was performed to confirm the fusion of IX with GFP gene in the genome of Ad5-IXGFP. Virions were incubated with suspended HEp-2 cells at 4 ℃ for 5 h, and the attachment of Ad5-IXGFP on the cellular membrane could be observed under fluorescence microscope. Conclusion GFP-labelled recombinant adenovirus was successfully constructed, which could be a valuable tool for subcellular localization fo pIX after virus entry real-time single adenovirus tracking by confocal fluorescence microscopy. Key words: Adenovirus; capsid protein IX; GFP; fluorescent labeling