title: Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.
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 time-consuming 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 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).
authors.affiliation: Department of Cardio-Thoracic Surgery, Zhongshan City People Hospital, Zhongshan, Guangdong, China.
authors.forename: J M
authors.initials: JM
authors.lastname: Zheng
authors.affiliation: Department of Cardio-Thoracic Surgery, Hospital of Traditional Chinese Medicine of Zhongshan, Guangdong, China.
authors.forename: J L
authors.initials: JL
authors.lastname: Cui
authors.affiliation: Department of Endocrinology, Tongji Hospital, Tongji Medical College, Hua zhong University of Science and Technology, Wuhan, China.
authors.forename: W T
authors.initials: WT
authors.lastname: He
authors.affiliation: Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China.
authors.forename: D W
authors.initials: DW
authors.lastname: Yu
authors.affiliation: Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China.
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.affiliation: Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China.
authors.forename: Z K
authors.initials: ZK
authors.lastname: Chen
authors.affiliation: Department of Cardio-Thoracic Surgery, Tongji Hospital, Tongji Medical College, Hua zhong University of Science and Technology, Wuhan, China.
authors.forename: H M
authors.initials: HM
authors.lastname: Zhou
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051379:Mice; D011971:Receptors, Immunologic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C534712:TIGIT protein, human; D049452:Green Fluorescent Proteins