Cloning and expression of interferon-α/γ from a domestic porcine breed and its effect on classical swine fever virus

To further evaluate the clinical impact of recombinant PoIFN-α/γ, PoIFN-α/γ genes from a Chinese domestic big-white porcine breed were cloned using PCR, and expressed in a high-level prokaryotic system. The antiviral activities of rPoIFN-α/γ on vesicular stomatitis virus (VSV), porcine reproductive and respiratory syndrome virus (PRRSV), and classical swine fever virus (CSFV) were investigated in different cell lines. The cloned PoIFN-α gene encodes a protein of 166 amino acids and has been named PoIFN-αc. In a comparison of PoIFN-αc with reported PoIFN-αI genes, eight amino acid substitutions at positions 43 (F to L), 78 (N to D), 86 (Y to C), 104 (A to V), 118 (R to L), 128 (T to P), 151 (S to V), and 156 (R to T) were observed, and resulted in no potential N-glycosylation site in the deduced PoIFN-α amino acid sequences. In contrast to PoIFN-αc, one nucleotide substitution was found at position 462 (A to G), hence 0.1% synonymity is specific for the PoIFN-γ gene. Both PoIFN-αc and PoIFN-γ genes were inserted into a prokaryotic vector pQE30, and expressed in E. coli M15 (pREP4) or SC11103 (pREP4) with the N-terminal six consecutive histidine residues, respectively. rPoIFN-αc and rPoIFN-γ proteins were detected by SDS-PAGE and Western blotting analysis at 20.7 and 18.0 kDa, respectively. In addition, the rPoIFN-αc and rPoIFN-γ protein were purified using Ni–NTA metal-affinity chromatography, and their anti-VSV, anti-PRRSV, and anti-CSFV activities were surveyed in homologous and heterologous cell lines. The results suggested that rPoIFN-α and rPoIFN-γ could inhibit classical swine fever virus and other important viral pathogens in different cell lines.