The production and biological assessment of cervine interferon gamma.
Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Escherichia coli expression system pET-32. The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to the IFN-gamma molecule. The ability of IFN-gamma to inhibit the killing of Madin-Darby bovine kidney cells by Semliki forest virus was used as a measure 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 model, it was shown that cervine rIFN-gamma was able to down-regulate the transcription of interleukin 10 mRNA while up-regulating the transcription of interleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood mononuclear cells. A prototype ELISA was tested for its ability to detect both recombinant 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 IFN-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.
Slobbe
L
L
Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
Lockhart
E
E
Kelly
J
J
Buchan
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D003670:Deer; D004797:Enzyme-Linked Immunosorbent Assay; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear; D009163:Mycobacterium bovis; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014376:Tuberculosis / Q000378:metabolism / Q000662:veterinary
D000911:Antibodies, Monoclonal; D007376:Interleukin-2; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D016753:Interleukin-10; D007371:Interferon-gamma