title: A simplified system for generating recombinant E3-deleted canine adenovirus-2.
abstract: Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector.
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Zuo
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Jiasen
authors.initials: J
authors.lastname: Liu
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Dongchun
authors.initials: D
authors.lastname: Guo
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Chuansong
authors.initials: C
authors.lastname: Quan
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Botao
authors.initials: B
authors.lastname: Li
authors.affiliation: Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China. Electronic address: yuzuosy@163.com.
authors.forename: Liandong
authors.initials: L
authors.lastname: Qu
mesh_terms: D006516:Adenoviruses, Canine / Q000235:genetics*; D000818:Animals; D003588:Cytopathogenic Effect, Viral; D004285:Dogs; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Canine adenovirus type 2; Enhanced green fluorescent protein; Homologous recombination; Shuttle vector; Vector