title: Cloning, expression and characterization of biologically active feline tumour necrosis factor-alpha.
abstract: We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-alpha (fTNF-alpha). Messenger RNA was extracted from feline peritoneal macrophage cultures and used to synthesize cDNA for polymerase chain reaction (PCR) amplification. The PCR products were cloned into the plasmid vector pCRII and sequenced, showing 99.3% homology with a published fTNF-alpha gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-alpha and glutathione S-transferase (GST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-alpha antibody in Western blotting, but not with a polyclonal anti-murine TNF-alpha serum. Recombinant fTNF-alpha (rfTNF-alpha) and rfTNF-alpha-GST had a CD50 of 15 ng ml-1 and 230 ng ml-1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-alpha-GST intravenously manifested the typical biological effects of TNF-alpha, including fever, depression, and piloerection. The rfTNF-alpha-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-alpha receptor and MHC-I antigen expression.
authors.affiliation: Department of Medicine, School of Veterinary Medicine, University of California, Davis 95616, USA.
authors.forename: E
authors.initials: E
authors.lastname: Rimstad
authors.forename: G H
authors.initials: GH
authors.lastname: Reubel
authors.forename: G A
authors.initials: GA
authors.lastname: Dean
authors.forename: J
authors.initials: J
authors.lastname: Higgins
authors.forename: N C
authors.initials: NC
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002371:Cat Diseases / Q000209:etiology; D002415:Cats; D003001:Cloning, Molecular; D003429:Cross Reactions; D003602:Cytotoxicity, Immunologic; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005334:Fever / Q000209:etiology / Q000662:veterinary; D015870:Gene Expression; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011817:Rabbits; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013047:Specific Pathogen-Free Organisms; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D000949:Histocompatibility Antigens Class II; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha