Cloning，Sequence Analysis And Expression On Porcine IFIT2/5Genes

The IFIT family, also named ISG56family, is a class of interferon-inducible genes andcomposed of4members, IFIT1(also named ISG56), IFIT2(also named ISG54), IFIT3(alsonamed ISG60) and IFIT5(also named ISG58). All of these IFIT members are clustered in a locuson the same chromosome and composed of only2exons separated by an intron of a few kilobasesin length. Their transcription is strongly induced by virus infection, interferons,lipopolysaccharides, and so on. These proteins were characterized as inhibitors of cellular as wellas viral processes, including translation, signaling, cell migration, proliferation, and viralreplication. In the present study, the complete coding sequence (CDS) of porcine IFIT2and IFIT5genes were cloned using molecular biology techniques, their polymorphisms in exon2wereidentified using direct PCR sequencing method, and the expression profiles were investigated withquantitative real-time PCR as well. Also, major regulatory regions and potential transcriptionfactors were characterized in porcine IFIT2promoter. Furthermore, the anti-disease molecularmechanism of porcine IFIT2and IFIT5was investigated, preliminarily. The main results are asfollowings:1. The cDNA of porcine IFIT2obtained was1558bp in length containing the complete CDSof1407bp. The CDS region is composed of two exons with the first exon providing the first fivenucleotides of the CDS, and the second exon containing the remaining part. Conceptual translationpredicted that IFIT2protein was composed of468amino acids. The sequence has been submittedto the GenBank database under accession No. JX070559.2. The cDNA of porcine IFIT5obtained was1558bp in length containing the complete CDSof1449bp. The CDS region is composed of two exons with the first exon providing the first fivenucleotides of the CDS, and the second exon containing the remaining part. Conceptual translationpredicted that IFIT5protein was composed of482amino acids. The sequence has been submittedto the GenBank database under accession No. JX280459.3. Five SNPs, c.259G>A、c.520T>G、c.571C>T、c.879A>G and c.889A>G, were found inporcine IFIT2gene,4of which resulting in amino acid substitution (p.Gly87Ser、p.Phe174Val、p.Pro191Ser and p.Ala297Thr). SNPs c.259G> A and c.520T> G may have an impact on proteinconformation and function by bioinformatics analysis.4. Porcine IFIT5gene was much conservative, in that only one synonymous SNP, c.462A>G(p.K154K), was found in exon2.5. The transcripts of both genes were unevenly distributed in all tissues studied, including heart, bladder, liver, large intestine, spleen, small intestine, lung, kidney, stomach, muscle, andlymph. IFIT2transcribes abundantly in spleen, liver, and heart, while IFIT5transcribes abundantlyin liver, heart, and small intestine.6. Minimal promoter region of porcine IFIT2exists within positions-162and-126bp.Negative regulatory regions were characterized in positions-1838~-1759and-389~-310bp,while positive regulatory regions in positions-1661~-1579,-310~-225and-225~-162bp.Bioinformatics analysis revealed ISRE binding factor, NF-kappaB, Delta EF1，c-Rel，and Id3maybe the important transcription factors for porcine IFIT2transcription.7. The over-expression of porcine IFIT2and IFIT5did not obviously affect the expression ofluciferase reporter gene regulated by ISRE. IFIT5had negative regulatory effects on the activationof NF-kappa B, as revealed by dual-luciferase reporter gene analysis.