Cloning and high level expression of bovine interferon gamma gene in eukaryotic cells [COS-7]

ABSTRACT

Interferon gamma [IFN-gamma] is one of the key cytokines in defining T helper 1 lymphocyte immune responses. In this study, the bovine IFN-gamma gene was cloned from spleen tissue RNA using the reverse transcription-polymerase chain reaction [RT-PCR]. IFN-gamma cDNA was sub-cloned and expressed in mammalian expression plasmid [pcDNA3.1[+]] under the control of the human cytomegalovirus [CMV] promoter. The predicted amino acid [aa] sequence of bovine IFN-gamma compared with corresponding known sequence from bovine [Bos taurus] was 100% identity and with ovine, caprine, camel, lama, equine, canine, feline, human, mice and chicken cytokine was 95, 95, 86, 83, 77, 75, 75, 61, 44 and 35%, respectively. Invitro expression of recombinant bovine IFN-gamma [rBoIFN-gamma] and secretion to culture medium was confirmed by ELISA test. Maximum expression of rBoIFN-gamma occurred at 96 and 144 h after transfection in COS-7 cells. These results showed that pcDNA3.1 expression vector and COS-7 cells transfected by diethylaminoethyl [DEAE]-dextran allowed the high level expression of bovine IFN-gamma gene and the release of protein in supernatant of cell culture