Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-? Proteins

Wang, Xiaona; Li, Fengsai; Han, Meijing; Jia, Shuo; Wang, Li; Qiao, Xinyuan; Jiang, Yanping; Cui, Wen; Tang, Lijie; Li, Yijing; Xu, Yi-Gang

In this study, the characteristics of the feIFN-ωa and feIFN-ωb proteins, such as signal peptide sequences, signal peptide cleavage sites, phosphorylation sites, glycosylation sites, antigen epitopes, hydrophobicity, and transmembrane regions were analyzed using bioinformatics to provide better theoretical guidance for the functional study of these proteins. The mature proteins, with normal biological activity, were formed only after the signal peptide sequence was removed from the precursor protein, thus allowing them to be secreted outside the cell membrane [28, 29] . We used online software to predict that the signal peptide sequence of the feIFN-ωa/ωb proteins consists of 23 amino acid residues, and that the signal peptide cleavage site is located between residues Gly23 and Cys24. The results indicated that the recombinant feIFN-ωa and feIFN-ωb proteins could be expressed in vitro in their soluble forms. Glycosylation is an important post-translational modification process that can affect the antigenic determinants, charge properties, enzymatic properties, and thermal stability of proteins. Similar to previous reports for the 13 known feIFN-ω subtypes [12] , we observed no N-glycosylation sites present in feIFN-ωa or feIFN-ωb. However, our analyses predicted nine potential O-glycosylation sites in feIFN-ωa and six potential O-glycosylation sites in feIFN-ωb, which is different from previous reports on the other known IFN-ω subtypes [6] . Studies have shown that glycosylation sites can play an important role in determining the activity of IFNs [30, 31] . For example, glycosylated IFN-ω has been observed to be markedly more potent than non-glycosylated IFN-ω against hepatitis C virus, BVDV, yellow fever virus, and West Nile virus, with even more superior effects than IFN-α, IFN-β, and IFN-γ [6, 30] .