Cloning and expression in mammalian cells of porcine tumor necrosis factor alpha: examination of biological properties.
We have cloned a full length complementary DNA (cDNA) of the porcine tumor necrosis factor alpha (pTNF-alpha) gene and expressed it in porcine and murine cells. Total RNA obtained from lipopolysaccharide (LPS) stimulated porcine peripheral blood mononuclear cells was reverse transcribed with a specific antisense pTNF-alpha primer to generate a single stranded cDNA which was subsequently amplified by the polymerase chain reaction utilizing an additional pTNF-alpha specific sense primer. The resulting double stranded cDNA was introduced into the pBMGNeo expression vector and transfected by electroporation in porcine (PK(15)) and murine (L929) cell lines. TNF-alpha bioactivity was detected in the supernatant of the transfected cells using a standard L929 bioassay or a PK(15) bioassay. The activity was zinc inducible as expected for a gene controlled by a metallothionein promoter. The bioactivity was not lowered by an anti-mouse TNF-alpha antiserum neutralizing murine, but not human TNF-alpha and a broad immunoreactive band of 17-19 kD was detected using an anti-mouse TNF-alpha serum suitable for immunoblotting. This newly developed tool will allow us to investigate the role of TNF-alpha in pathogenesis of viral infections and gram-negative sepsis.
Von Niederhäusern
B
B
Institute of Veterinary Virology, University of Bern, Switzerland.
Bertoni
G
G
Hertig
C
C
Pfister
H
H
Peterhans
E
E
Pauli
U
U
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004247:DNA / Q000235:genetics; D015151:Immunoblotting / Q000662:veterinary; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000276:immunology; D013552:Swine / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA