In Vitro Transgenic Expression Efficacy Of A Helper-dependent Adenoviral Vector Encoding Enhanced Green Fluorescent Protein

Object: Helper-dependent adenoviral vector (HDAd) was mainly used in gene therapy research. Currently, two papers of HDAd as systemic vaccine vector have been reported, which demonstrate HDAd can generate greater cellular and humoral immune responses against transgene than first generation adenoviral vector (FGAd). Generally, such enhanced immune responses have been attributed to the elevated expression level of transgene and the attenuated host immune response induced by HDAd in vivo, as compaerd with FGAd. It has fewer reports about in vitro trangenic expression efficiency of these two adenoviral vectors. By absorbing blue light, green fluorescent protein (GFP) can emit green light. The light-stimulated GFP fluorescence is species independent, free of co-factors or substrates and non-toxic. Therefore, it has been widely used as a reporter in many gene delivery technologies. Enhanced green fluorescent protein (EGFP) is a modified edition of GFP with optimized codons for human and amino acid substitution for brighter fluorescence and higher expression in mammalian cells. In order to investigate in vitro transgenic expression efficiency, we constructed a HDAd encoding EGFP (HADd/EGFP), performed large scale preparation and purification, and then compared the transgenic expression efficiency of HDAd/EGFP with FGAd/EGFP in vitro.Methods: We amplified the expression cassette of EGFP including CMV promoter, open reading frame (ORF) of EGFP gene and SV40 polyA by polymerase chain reaction, which was subcloned into a shuttle vector of pSC11 and then into a backbone vector of pSC15B. We liberated HDAd/EGFP genome from the resulting recombinant vector pSC15B/EGFP with restriction enzyme Pmeâ… , and then transfected the linear HDAd/EGFP DNA into 293Cre4 cells with calcium phosphate transfection method. The 293Cre4 cells were infected by helper virus H14 16 hours after transfection. HDAd/EGFP was amplified by serial coinfection of 293Cre4 cells by H14 and the crude lysates until it reached the plateau of amplification. HDAd/EGFP was purified by CsCl gradient ultracentrifugation and identified under fluorescent microscope and electron microscope. The concentration of HDAd/EGFP particles in the purified preparation was determined by spectrophotometer and expressed as virus particle (vp). A549 cells were infected by 2000 vp per cell by HDAd/EGFP and FGAd/EGFP respectively, and analyzed by flow cytometry.Result: HDAd/EGFP was successfully constructed,prepared and purified. After infecting 293Cre4 cells by purified HDAd/EGFP, the expression of EGFP was confirmed under fluorescent microscope and typical adenoviruses morphology was observed under electron microscope. Flow cytometry was exploited to determine EGFP expression efficiency by analyzing A549 cells infected with same amount of HDAd or FGAd, and the results showed that the fluorescent expression rate and fluorescent intensity of EGFP were higher in infected A549 cells by HDAd/EGFP than by FGAd/EGFP.Conclusion: HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.