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.
Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
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L
Slobbe
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Lockhart
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J
Kelly
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Buchan
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