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 t umor necrosis factor a (pTNF-alpha) gene and expressed it in porcine a nd murine cells. Total RNA obtained from lipopolysaccharide (LPS) stim ulated porcine peripheral blood mononuclear cells was reverse transcri bed with a specific antisense pTNF-alpha primer to generate a single s tranded cDNA which was subsequently amplified by the polymerase chain reaction utilizing an additional pTNF-alpha specific sense primer . Th e resulting double stranded cDNA was introduced into the pBMGNeo expre ssion vector and transfected by electroporation in porcine (PK(15)) an d 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 no t lowered by an anti-mouse TNF-alpha antiserum neutralizing murine, bu t not human TNF-alpha and a broad immunoreactive band of 17-19 kD was detected using an anti-mouse TNF-alpha serum suitable for immunoblotti ng. This newly developed tool will allow us to investigate the role of TNF-alpha in pathogenesis of viral infections and gram-negative sepsi s.