Cloning and Expression of the Canine Interferon-γ Gene

Abstract

The canine interferon-γ (cIFN-γ) chromosomal gene was isolated from a recombinant library, containing the entire dog genome, by screening with a rat IFN-γ genomic probe. The structural organization of the cIFN-γ gene closely resembles that of the human, murine, and rat IFN-γ genes. It contains three intervening sequences and encodes a signal sequence of 23 amino acids followed by a mature protein of 143 amino acids. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the cIFN-γ protein sequence with that of the corresponding murine, rat, human, and bovine proteins revealed a homology of 40%, 42%, 65%, and 76%, respectively. The cIFN-γ gene was expressed under control of the simian virus 40 early promoter in Chinese hamster ovary (CHO) cells, deficient in dihydrofolate reductase (DHFR), after cotransformation with a plasmid containing the murine dhfr gene. Initial transformants with dhfr⁺ phenotype produced cIFN-γ titers ranging from 10 to 10,000 laboratory units (LU)/ml of culture medium. A cIFN-γ cDNA sequence was identified in a cDNA library constructed with partially purified RNA from a cIFN-γ– producing CHO cell line. In vitro transcription of the cDNA and translation of the mRNA in Xenopus laevis oocytes resulted in secreted IFN-γ with an antiviral activity specific for canine cells.