title: Assessment of bioactivity of a recombinant chicken interferon-gamma expressed using a baculovirus expression system.
abstract: The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1β, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-β chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.
authors.affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Canada.
authors.forename: Amirul I
authors.initials: AI
authors.lastname: Mallick
authors.forename: Kamran
authors.initials: K
authors.lastname: Haq
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Brisbin
authors.forename: M Firoz
authors.initials: MF
authors.lastname: Mian
authors.forename: Raveendra R
authors.initials: RR
authors.lastname: Kulkarni
authors.forename: Shayan
authors.initials: S
authors.lastname: Sharif
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D005240:Feasibility Studies; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics
chemical_list: D030161:Avian Proteins; D016207:Cytokines; D000949:Histocompatibility Antigens Class II; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II