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. 2006 Jan;29(1):19-24.

Expression of bioactive recombinant bovine interferon-gamma using baculovirus

Giovanna Gentilomi  1 , Rossella Lelli, Mirella D'Angelo, Vincenzo Langella, Federica Monaco, Ottavio Portanti, Mirella Luciani, Mara Mirasoli, Aldo Roda, Marialuisa Zerbini, Monica Musiani
Affiliations
  • PMID: 16608121

Expression of bioactive recombinant bovine interferon-gamma using baculovirus

Giovanna Gentilomi et al. New Microbiol. 2006 Jan.

Abstract

The precise role of bovine interferon-gamma (BoIFN-gamma) in disease and therapy is still poorly defined. Clearly it is involved in defence against parasites, bacteria, viruses and possibly tumor cells. This paper reports the expression of BoIFN-gamma in a baculovirus system to generate a fully functional recombinant protein. Bovine interferon-gamma cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) encoding for a putative 166 amino acid protein (22KDa) was cloned and expressed into baculovirus transfer vector pBlueBac 4.5/V5 His. This vector was co-transfected with Autografa californica multiple nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugiperda cells (Sf9) and the recombinant virus, named AcBoIFN-gamma, was then recovered. Recombinant BoIFN-gamma (rBoIFN-gamma His) was accumulated in the serum-free medium of AcBoIFN-gamma-infected cells. The nickel affinity spin column purified rBoIFN-gamma His was shown to be a glycosylated 20-22 KDa protein as confirmed by SDS-PAGE glycan determination and showed antiviral activity in vitro against the bovine viral diarrhoea-mucosal disease virus (BVD/MD). The production of this bioactive rBoIFN-gamma His will allow us to explore this cytokine as a potential vaccine adjuvant or therapeutic agent for bovine diseases.

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