Cloning, expression and characterization of biologically active feline tumour necrosis factor-α

Espen Rimstad, Gerhard H. Reubel, Gregg A. Dean, Joanne Higgins, Niels C Pedersen

Research output: Contribution to journalArticle

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Abstract

We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (fTNF-α). 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-α gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-α 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-α antibody in Western blotting, but not with a polyclonal anti-murine TNF-α serum. Recombinant fTNF-α (rfTNF-α) and rfTNF-α-GST had a CD50 of 15 ng ml-1 and 230 ng ml-1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-α-GST intravenously manifested the typical biological effects of TNF-α, including fever, depression, and piloerection. The rfTNF-α-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-α receptor and MHC-I antigen expression.

Original languageEnglish (US)
Pages (from-to)297-310
Number of pages14
JournalVeterinary Immunology and Immunopathology
Volume45
Issue number3-4
DOIs
StatePublished - 1995

Fingerprint

tumor necrosis factors
Felidae
Organism Cloning
molecular cloning
Glutathione Transferase
Tumor Necrosis Factor-alpha
cats
glutathione transferase
Proteins
Piloerection
proteins
Polymerase Chain Reaction
polymerase chain reaction
Tumor Necrosis Factor Receptors
Interleukin-2 Receptors
antigens
Peritoneal Macrophages
plasmid vectors
receptors
thrombin

ASJC Scopus subject areas

  • Immunology
  • veterinary(all)
  • Animal Science and Zoology

Cite this

Cloning, expression and characterization of biologically active feline tumour necrosis factor-α. / Rimstad, Espen; Reubel, Gerhard H.; Dean, Gregg A.; Higgins, Joanne; Pedersen, Niels C.

In: Veterinary Immunology and Immunopathology, Vol. 45, No. 3-4, 1995, p. 297-310.

Research output: Contribution to journalArticle

Rimstad, Espen ; Reubel, Gerhard H. ; Dean, Gregg A. ; Higgins, Joanne ; Pedersen, Niels C. / Cloning, expression and characterization of biologically active feline tumour necrosis factor-α. In: Veterinary Immunology and Immunopathology. 1995 ; Vol. 45, No. 3-4. pp. 297-310.
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