title: Generation of a replication-deficient recombinant human adenovirus type 35 vector using bacteria-mediated homologous recombination.
abstract: The use of adenovirus type 35 (Ad35) as a vector in vaccine and gene therapy studies is promising due to its broad cell tropism and low seroprevalence in humans. However, to date, a simple and effective system for producing recombinant Ad35 (rAd35) has not been well developed. This report describes a two-plasmid Ad35-Easy system to facilitate the production of recombinant Ad35 (rAd35). The system employed the pAd35-shuttle vector for foreign gene transfer and the pAd35-backbone vector to provide the Ad35 genomic backbone. A 293-Ad35E1B cell line was used to trans-complement rAd35 replication. rAd35 plasmids were obtained through homologous recombination following co-transformation of E. coli BJ5183 cells with recombinant pAd35-shuttle vectors harboring foreign genes. rAd35 viruses were obtained directly by transfecting 293-Ad35E1B cells with foreign gene-containing rAd35 plasmids and the pAd35-backbone vector. The production of E1 deficient rAd35 was evaluated by transfecting the 293-Ad35E1B cells with the rAd35 plasmid containing the enhanced green fluorescent protein (EGFP) gene. The virus grew effectively at a yield comparable to that of wild type Ad35 in HEp2 cells, indicating that the Ad35-Easy system is an efficient method for rapid production of rAd35 in sufficient quantities for vaccine development or gene therapy.
authors.affiliation: State Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Chengjun
authors.initials: C
authors.lastname: Wu
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Lei
authors.forename: Jianwei
authors.initials: J
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Hung
mesh_terms: D017501:Adenovirus E1B Proteins / Q000235:genetics / Q000378:metabolism; D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D045744:Cell Line, Tumor; D003673:Defective Viruses / Q000235:genetics* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics*; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D019065:Virus Assembly; D014779:Virus Replication / Q000235:genetics*
chemical_list: D017501:Adenovirus E1B Proteins