Construction of human adenovirus type 4 vector expressing enhanced green fluorescence protein

Objective To prepare human adenovirus type 4 (Ad4) vector expressing enhanced green fluorescence protein (EGFP). Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombinant plasmid was transfected into AD293 cells to rescue recombinant virus which was identified by sequencing, SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP expressing EGFP was obtained and could be recognized and neutralized by monoclonal antibody MN4b and antisera against Ad4. The Ad4-specific and EGFP-specific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development, drug evaluation and transgene vector. Key words: Human adenovirus type 4; Vector; Enhanced green fluorescence protein; Neutralization