Sequencing characterization and expression analysis of interferon gamma (IFN-γ) in Rusa deer (Rusa timorensis) and Thai cervidae

Abstract

To date, the interferon gamma (IFN-γ) assay has become an important diagnostic tool which is applied alternatively to define infection of intracellular pathogens. In this study, the molecular characteristics of IFN-γ in Rusa deer (Rusa timorensis), Hog deer (Axis porcinus), Sambar deer (Rusa unicolor) and Eld’s deer (Rucervus eldii) were determined. Among the cervid family, the analysis of IFN-γ cDNA sequences indicated 5 polymorphism nucleotide positions. Deduced IFN-γ amino acid compositions among the cervid group were negligibly different (98-100% homology). Preliminary quantification of IFN-γ mRNA expression in the cervidae by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was demonstrated. qPCR primers were designed and tested for their ability to quantify IFN-γ mRNA with a suggested detection limit at 103 target molecules. Intra- and inter-assay variations of RT-qPCR for IFN-γ were 1.41 and 4.57, respectively. Expression ratio of IFN-γ mRNA in mitogen-stimulated peripheral blood mononuclear cells (PBMCs) relative to non-stimulated cells after normalization against β2-microglobulin (β2M) in the Rusa deer, Eld’s deer and Hog deer were 6.47±3.39, 22.91±7.21 and 17.66±1.30, respectively. This preliminary demonstration may serve as the basic instrument for further development of rapid, simple and reliable immunodiagnostic methods for the study of cervidae in Thailand.