Cloning, expression and bioactivity of porcine soluble TNF receptor 1 and the comparison of PK(15) and WEHI 164(13)-based TNF bioassays

摘要

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that activates both leukocytes and endothelium and facilitates the movement of inflammatory cells from circulation to sites of infection or injury. This activation may be beneficial and aid in host defense, or may be detrimental and mediate part of the pathophysiology of disease. Several porcine diseases, such as salmonellosis and mycoplasmal pneumonia, cause an increase in TNF production, which may be either beneficial or harmful to swine health. Two related studies were completed to develop assays and reagents to further study the role of TNF in porcine disease;In the first study, we compared the sensitivity of PK(15) and WEHI 164(13) cells to human, murine and porcine TNF-mediated lysis. Our data indicated that the PK(15) cells are 50 times less sensitive to murine TNF and 15-fold less sensitive to human TNF than are WEHI 164(13) cells. The PK(15) cells are, however, 4 times more sensitive to recombinant porcine TNF and 15 times more sensitive to porcine serum containing TNF. Because the PK(15)-based bioassay was more sensitive for detecting porcine TNF in serum, this bioassay may be particularly useful in the study of infectious disease processes of swine;In mice and humans, sepsis and endotoxemia are also accompanied by a rise in circulating soluble receptors for TNF. Since there is little known about these receptors during porcine sepsis, the objectives of the second study were to clone, express and determine the bioactivity of porcine soluble TNF receptor 1 (TNFR1). Using a polymerase chain reaction (PCR)-based library 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. An anti-FLAG affinity column was used to purify the fusion protein. The purified protein at a concentration of 5 [mu]g/ml neutralized 70% of TNF-mediated cytotoxicity in a bioassay. Recombinant porcine soluble TNFR1 protein and the PK(15) based bioassay may be useful in studying the roles of both TNF and TNFR1 in the pathogenesis of infectious disease of swine.