Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E. coli.

Abstract

We have utilized the reverse transcription polymerase chain reaction (RT-PCR) to clone the protein coding region of canine tumor necrosis factor (TNF-alpha) cDNA. The gene displays 90% sequence homology to the corresponding human TNF-alpha cDNA. The predicted initial translation product is 233 amino acids and shows 88% homology to the human counterpart, and 92% homology with the human putative mature TNF-alpha protein. The canine TNF-alpha clone was used to engineer bacteria to express large amounts of the mature form of recombinant protein. A monoclonal antibody against human TNF-alpha cross-reacted with canine rTNF-alpha using Western blot and ELISA analysis. The purified canine rTNF-alpha had a cytotoxic effect on WEHI 164 clone 13 cells as well as increasing the cell surface expression of major histocompatibility class II antigens on canine kidney cortical cell line (MDCK) in vitro. The availability of canine rTNF-alpha will allow further studies on its role in immunoregulatory mechanisms in the canine transplantation model, both by itself and in conjunction with the already available canine specific recombinant interferon-gamma.