Molecular characteristics and tissue expression analysis of chicken interferon regulatory factor 7

【Objective】 The aim of this study was to analyze the molecular characteristics of chicken interferon regulatory factor 7(chIRF7) and the expression characteristics of chIRF7 gene in different chicken tissues at different developmental stages, which provided a foundation for further investigating the role of chIRF7 gene in regulating chicken tissue development and antiviral study.【Method】 The coding DNA sequence(CDS) of chIRF7 gene were amplified by RT-PCR and then used to construct the recombinant eukaryotic expression vector. Bioinformatics online software, including SOPMA, SWISS-MODEL and BioEdit, were performed to analyze the secondary and tertiary structures and conserved domains of chIRF7 protein. In addition, chicken embryo fibroblast cell lines(DF-1) transfected withthe recombinant eukaryotic expression vector were used to observe the subcellular localization of recombinant chIRF7, and study the effect of fusion protein overexpression on the replication of Newcastle disease virus(NDV). Moreover, real-time fluorescencequantitative PCR was performed to detect the expression of chIRF7 gene in different tissues at 14-day-old(E14d) chicken embryos, and at 1(H1d), 7(H7d), and 14 d(H14d) after hatching.【Result】 The CDS region of chIRF7 gene was amplified from the antisense transcript derived from total RNA of chicken peripheral blood lymphocytes, which was inserted into pEGFP-C1 to construct the recombinant eukaryotic expression vector pEGFP-chIRF7. The secondary structures of chIRF7 were composed of irregular coil(51.12%), α-helix(26.48%), extended chain(16.70%), and β-turn(5.70%). IRF7 amino acid of chicken had low homology with other avian species, human and mammals, and the five functional domains were not conserved among them. The subcellular localization of fusion protein EGFP-chIRF7 changed from the cytoplasm to the nucleus in DF-1 cells treated with polyinosinic-polycytidylic acidPoly(I:C)or NDV. Meanwhile, fusion protein EGFP-chIRF7 overexpression reduced the replication ability and cytopathogenicity of NDV. chIRF7 gene was expressed in different chicken tissues at different developmental stages, and the relative expression of chIRF7 gene was the highest in lung, followed by liver and glandular stomach, while the relative expression of chIRF7 gene was low in leg muscle. From E14d to H14d, the expression of chIRF7 gene in lung had a trend of decrease first and then stabilization, while it showed decrease first, then increase and decrease again in liver, glandular stomach, heart and leg muscle. As for brain tissue, the expression of chIRF7 gene was initially stabilized and then decreased, however, the expression in pectoral muscle and eyeball tended to be stable.【Conclusion】 The similarity of IRF7 in different species is low, and the domain is not conserved, and chIRF7 expression induced by external stimulation shows nuclear translocation along with antiviral infection effect. IRF7 gene has the highest expression in chicken lung, liver and glandular stomach at different developmental stages, indicating the potential regulatory role in the development of these three tissues.