Expression of bioactive recombinant bovine interferon-gamma using baculovirus.
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.
Gentilomi
Giovanna
G
Department of Clinical Experimental Medicine, Microbiology Section, University of Bologna, Italy. giovanna.gentilomi@unibo.it
Lelli
Rossella
R
D'Angelo
Mirella
M
Langella
Vincenzo
V
Monaco
Federica
F
Portanti
Ottavio
O
Luciani
Mirella
M
Mirasoli
Mara
M
Roda
Aldo
A
Zerbini
Marialuisa
M
Musiani
Monica
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D012313:RNA; D007371:Interferon-gamma