Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene

Abstract

ABSTRACT. Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and timeconsuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression 18651©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 18650-18661 (2015) Construction and characterization of TIGIT-GFP adenovirus must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10 10 PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

Recombinant Human Adenovirus Type 5 Co-expressing RABV G and SFTSV Gn Induces Protective Immunity Against Rabies Virus and Severe Fever With Thrombocytopenia Syndrome Virus in Mice

“…The Ad5 was used as the vector to express target proteins. The construction of the recombinant Ad5-G-Gn carrying the RABV (SRV9 strain) G and SFTSV (JS-2011-013-1 strain) Gn coding sequence, linked by P2A gene, was conducted, as previously described (Zheng et al, 2015). Schematic diagram of the Ad5-G-Gn was shown in Figure 1A.…”