Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/13/2001
Publication Date: N/A
Technical Abstract: Tumor necrosis factor is an important mediator of inflammation and septic shock. We have previously described the production in the yeast Pichia pastoris of a recombinant soluble bovine tumor necrosis factor receptor type I that potently neutralized bovine tumor necrosis factor. Using PCR methodology, we have generated a construct encoding the signal and extracellular domain of the bovine tumor necrosis factor receptor type I fused to the hinge and constant domains 2 and 3 of the heavy chain of bovine IgG1. The sequence was placed under the transcriptional control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) by site-specific transposition and the encoded protein expressed in Sf21 fall armyworm cells and purified by a single-step protein G affinity chromatography. As much as 4 mg of purified protein were obtained from a liter of starting culture using this system. In in vitro assays, recombinant soluble bovine tumor necrosis factor receptor I-IgG1 fusion (rsboTNF-RI-IgG1) inhibited 50% and 99% of the cytotoxic activity of 1000 units of recombinant porcine TNFalpha on 4 x 10**3 WEHI 163 cells when used at concentrations of 3.1 and 6.7 picomoles/ml, respectively. In view of its strong TNF inhibitory activity, this receptor construct may prove useful as a therapeutic agent for the prevention and treatment of TNF-mediated inflammatory responses and sepsis in cattle.