CLONING, EXPRESSION AND CHARACTERIZATION OF BIOLOGICALLY-ACTIVE FELINE TUMOR-NECROSIS-FACTOR-ALPHA

We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-alpha (TNF-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. Sub cloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-alpha and glutathione S-transferase (G ST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-alpha ant ibody in Western blotting, but not with a polyclonal anti-murine TNF-a lpha serum. Recombinant fTNF-alpha (rfTNF-alpha) and rfTNF-alpha-GST h ad a CD50 of 15 ng ml(-1) and 230 ng ml(-1), respectively, in the L929 cytotoxicity assay. Cats given rfTNF-alpha-GST intravenously manifest ed the typical biological effects of TNF-alpha, including fever, depre ssion, and piloerection. The rfTNF-alpha-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells s timulated in vitro, but had no effect on TNF-alpha receptor and MHC-I antigen expression.