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

Document Type: Full paper (Original article)

Authors

1 Ph.D. Student in Biotechnology, Department of Pathobiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Fellow Member of Academy of Science of Iran, Tehran, Iran

4 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

5 Department of Parasitology, Pasteur Institute, Tehran, Iran

6 Department of Biotechnology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

Abstract

Interferon gamma (IFN-γ) is one of the key cytokines in defining T helper 1 lymphocyte immune
responses. In this study, the bovine IFN-γ gene was cloned from spleen tissue RNA using the reverse
transcription-polymerase chain reaction (RT-PCR). IFN-γ 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-γ 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-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 in
supernatant of cell culture.

Keywords