ARS | Publication request: CLONING, EXPRESSION AND BIOACTIVITY OF SOLUBLE PORCINE TNFR1

Submitted to: International Virtual Conference on Infectious Diseases of Animals
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 1997
Publication Date: N/A

Technical Abstract: Infections with Salmonella spp. cause an increase in circulating tumor necrosis factor (TNF) in mice, humans, and swine. In mice and humans salmonellosis is also accompanied by a rise in circulating soluble receptors for TNF. Since there is little known about these receptors durine porcine salmonellosis, the objectives of this study were to clone, express and determine the bioactivity of the porcine soluble TNF receptor I (TNFR1). Using a PCR-based enrichment technique, a 927 base pair fragment of porcine TNFR1 was isolated from a lung cDNA library. The mature extracellular domain consisting of 464 amino acids of porcine TNFR1 was expressed as a FLAG fusion protein in Escherichia coli with a yield of 120-150 mg per liter of culture. The molecular weight of this fusion protein was approximately 29 kDa, 85% of which was the extracellular domain of porcine TNFR1. An anti-FLAG affinity column was used to purify the fusion protein. The purified protein at a concentration of 5 mg/ml neutralized 70% of TNF-mediated cytotoxicity in a PK(15) based bioassay. This recombinant porcine soluble TNFR1 protein may be useful in studying the roles of both TNF and TNFR1 in the pathogenesis of infectious disease of swine, such as salmonellosis.