cloning and high level expression of bovine interferon gamma gene in eukaryotic cells (cos-7)
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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.
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Journal title:
iranian journal of veterinary researchPublisher: shiraz university
ISSN 1728-1997
volume 11
issue 2 2010
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