| When host infected viruses, bacteria, parasites and fungi, type I interferon have important effects in the innate and adaptive immune system through the induction of other mediators. Type I IFNs are important for host defence against viruses. In this work, bovine has 24 IFN-omega genes: 22 functional bovine interferon genes and 2 pseudogenes were identified in the bovine genome data base by BLAST scanning, which is a type I interferon.Subtype-specific primers for the 14 functional Bo IFN-ω members were designed by alignment of 22 functional Bo IFN-ω members and all the members were amplified by PCR from bovine liver genomic DNA. The 14 functional Bo IFN-ω members share 91.3%~97.2% identity at the nucleotide level and 84.4%~95.4% identity at the amino acid level. Although 13 functional Bo IFN-ω(Bo IFN-ω7 and Bo IFN-ω8) are translated into 195 amino acid(AA) prepeptides, Bo IFN-ω11 only has 186 AA owing to a 9 AA C-terminal deletion. All 14 functional Bo IFN-ω genes have putative signal peptides of 23 AA, so that their mature peptides comprise 173 and 163(Bo IFN-ω11) amino acids, respectively. Cysteines at positions1/99 and 29/139 of the mature peptides are predicted to form disulfide bonds. Through the analysis of the phylogenetic tree and sequence alignment, we select 8 Bo IFN-ω members in prokaryotic expression, and analysis its biological activity and the primary physicochemical characteristics. This study revealed that 8 Bo IFN-ω members have different antiviral activity which can be abrogated by PAb against Bo IFN-ω24. Bo IFN-ω13 had the highest antiviral activity, about 47.5 times that of Bo IFN-ω11, which had the lowest antiviral activity. 8 Bo IFN-ω members have the typical characteristics of type I interferon, which have stable at p H 2.0 or 65°C. The antiproliferation activity was determined in vitro, which found 8 Bo IFN-ω subtype members could inhibit cell proliferation. The mRNA levels of Mx1 genes in theMDBK cells were enhanced after treatment with 8 Bo IFN-ω subtype members by Real-time PCR.Bovine consensus interferon-omega(Co Bo IFN-ω) is a recombinant non-naturally occurring Bo IFN-ω. It was obtained by synthesizing a codon-optimized gene composed of the most frequently occurring amino acid at each position in the bovine omega interferon family and then expressing in Pichia pastoris. CoBo IFN-ω presented higher antiviral and antiproliferative activities than naturally occurring Bo IFN-ω, and the antiviral activity of Co BoIFN-ω is 4.6×107 U/mgå'Œ1.6×107 U/mg for inhibition of vesicular stomatitis virus(VSV) in Madin-Darby bovine kidney(MDBK) and porcine kidney cells(PK-15), respectively. Besides, Co Bo IFN-ω enhanced the mRNA levels of Mx1 and that Co Bo IFN-ω had a better induction effect than that of naturally occurring Bo IFN-ω. Furthermore, Co Bo IFN-ω was stable at p H 2.0 and 63 °C. Therefore, this study revealed that Co Bo IFN-ω presents the typical characteristics of type I interferon, and this method of gene engineering can facilitate research to enable the construction of proteins with high antiviral activity, which can be used for both study and industrial purposes. |
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