Editorial Type:
Physiology

Cloning and expression of porcine recombinant soluble tumor necrosis factor receptor 1

Nancy Maroushek Boury From the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Maroushek Boury, Taylor); and Enteric Diseases and Food Safety (Bosworth, Stabel) and Metabolic Diseases and Immunology Research (Kehrli) Units, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010.

Search for other papers by Nancy Maroushek Boury in
Current site
Google Scholar
PubMed Close
 MS
,
Brad T. Bosworth From the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Maroushek Boury, Taylor); and Enteric Diseases and Food Safety (Bosworth, Stabel) and Metabolic Diseases and Immunology Research (Kehrli) Units, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010.

Search for other papers by Brad T. Bosworth in
Current site
Google Scholar
PubMed Close
 DVM, PhD
,
Thomas J. Stabel From the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Maroushek Boury, Taylor); and Enteric Diseases and Food Safety (Bosworth, Stabel) and Metabolic Diseases and Immunology Research (Kehrli) Units, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010.

Search for other papers by Thomas J. Stabel in
Current site
Google Scholar
PubMed Close
 PhD
,
Marcus E. Kehrli Jr From the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Maroushek Boury, Taylor); and Enteric Diseases and Food Safety (Bosworth, Stabel) and Metabolic Diseases and Immunology Research (Kehrli) Units, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010.

Search for other papers by Marcus E. Kehrli Jr in
Current site
Google Scholar
PubMed Close
 DVM, PhD
, and
Michael J. Taylor From the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (Maroushek Boury, Taylor); and Enteric Diseases and Food Safety (Bosworth, Stabel) and Metabolic Diseases and Immunology Research (Kehrli) Units, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010.

Search for other papers by Michael J. Taylor in
Current site
Google Scholar
PubMed Close
 MA, Vet MB, D Phil
Free access

Abstract

Objective

To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1).

Procedure

A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay.

Results

A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 μg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner.

Conclusions

Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine. (Am J Vet Res 1998;59:1317–1322)

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 5952 5900 67
PDF Downloads 67 44 2
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Issue:
01 Oct 1998
Advertisement