The production and biological assessment of cervine interferon gamma

Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Esch erichia coli expression system pET-32, The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to t he IFN-gamma molecule. The ability of IFN-gamma to inhibit the killing of M adin-Darby bovine kidney cells by Semliki forest virus was used as a measur e of the bioactivity of the recombinant cervine IFN-gamma (rIFN-gamma). It was shown that the presence of the thioredoxin fusion partner 5' to the IFN -gamma molecule did not affect its biological activity. As in the mouse mod el, it was shown that cervine rIFN-gamma was able to down-regulate the tran scription of interleukin 10 mRNA while up-regulating the transcription of i nterleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood monon uclear cells. A prototype ELISA was tested for its ability to detect both r ecombinant and native IFN-gamma, The ELISA was able to detect rIFN-gamma at concentrations greater than 100 pg/ml, It was also used to detect native I FN-gamma produced by peripheral blood lymphocytes from Mycobacterium bovis infected or vaccinated deer after in vitro restimulation with antigen. The rIFN-gamma and the cervine IFN-gamma specific ELISA provide valuable tools with which to study important zoonotic infections in farmed and wild deer. (C) 2000 Academic Press.