A stable mammalian cell line expressing highly active bovine interferon-gamma (BoIFN-gamma) was generated using Flp recombinase-mediated integration. This recombinant 293 cell line (B1) efficiently secreted FLAG-tagged BoIFN-gamma protein into the culture supernatant, as determined by ELISA and Western blot. The recombinant BoIFN-gamma exhibited high anti-viral activity, suggesting that the 293 cells expressed BoIFN-gamma that structurally and biologically resembled the natural protein. Two monoclonal antibodies (mAbs) with high affinity for the 293 cell-expressed BoIFN-gamma were identified using this cell line, and these mAbs can be used for the development of diagnostic kits. Thus, this work demonstrates the successful generation of a 293 cell line that produces large quantities of highly active BoIFN-gamma and demonstrates its potential application in the research of bovine infectious diseases. (C) 2014 Elsevier Inc. All rights reserved.