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

Abstract

Interferon gamma (IFN-γ) is one of the key cytokines in defining T helper 1 lymphocyte immuneresponses. In this study, the bovine IFN-γ gene was cloned from spleen tissue RNA using the reversetranscription-polymerase chain reaction (RT-PCR). IFN-γ cDNA was sub-cloned and expressed inmammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus (CMV)promoter. The predicted amino acid (aa) sequence of bovine IFN-γ compared with corresponding knownsequence 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-γ (rBoIFN-γ) and secretion to culture medium was confirmed by ELISA test. Maximum expression of rBoIFN-γ 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-γ gene and the release of protein insupernatant of cell culture.