鸡干扰素调节因子7的分子特征及其组织表达特性分析

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

【目的】明确鸡干扰素调节因子7(chIRF7)的分子特征及chIRF7基因在不同发育阶段各组织中的表达特点,为后续开展IRF7基因调控鸡相关组织发育和抗病毒研究打下基础。【方法】利用RT-PCR扩增chIRF7基因编码区(CDS)并构建其重组真核表达载体,通过SOPMA、SWISS-MODEL及BioEdit等在线软件预测分析chIRF7蛋白的二、三级结构及结构域保守性;以重组真核表达载体转染鸡胚成纤维细胞系(DF-1),观察融合蛋白的亚细胞定位情况及分析过表达融合蛋白对新城疫病毒(NDV)复制的影响;采用实时荧光定量PCR检测chIRF7基因在鸡胚发育第14 d(E14d)及鸡出壳第1 d(H1d)、第7 d(H7d)和第14 d(H14d)不同组织中的表达情况。【结果】将从鸡外周血淋巴细胞中扩增获得的chIRF7基因CDS序列插入真核表达载体pEGFP-C1的酶切位点即成功构建获得重组真核表达载体pEGFP-chIRF7。chIRF7蛋白二级结构由无规则卷曲(占51.12%)、α-螺旋(占26.48%)、延伸链(占16.70%)和β-转角(占5.70%)组成;鸡与其他禽类、人类和哺乳动物的IRF7氨基酸序列相似性均较低,且5个功能结构域不保守。经聚肌胞苷酸[Poly(I∶C)]或NDV处理DF-1细胞后,可使融合蛋白EGFP-chIRF7由细胞质定位转变为细胞核定位;而在细胞内过表达融合蛋白EGFP-chIRF7可降低NDV的复制能力和细胞致病性。chIRF7基因在鸡不同发育阶段各组织中均有不同程度的表达,且以肺脏中的相对表达量最高,其次是肝脏和腺胃,在腿肌中的相对表达量较低;从E14d发育到H14d,chIRF7基因在肺脏中的表达呈先下降后趋于稳定的变化趋势,在肝脏、腺胃、心脏和腿肌中呈先下降后上升再下降的变化趋势,在脑组织中呈先稳定再下降的变化趋势,在胸肌和眼球中的表达则趋于稳定。【结论】IRF7在不同物种中的相似性较低且结构域不保守,经外界刺激诱导表达的chIRF7会发生细胞核移位而具有抗病毒感染效果。IRF7基因在鸡不同发育阶段的肺脏、肝脏和腺胃中高表达,说明对这3个组织的发育可能具有调控作用。

【Objective】The aim of this study was to analyze the molecular characteristics of chicken interferon regula-tory factor 7(chIRF7)and the expression characteristics of chIRF7 gene in different chicken tissues at different develop-mental 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 SOP-MA,SWISS-MODEL and BioEdit,were performed to analyze the secondary and tertiary structures and conserved do-mains of chIRF7 protein.In addition,chicken embryo fibroblast cell lines(DF-1)transfected withthe recombinant eu-karyotic 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 fluorescence-quantitative 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 ampli-fied from the antisense transcript derived from total RNA of chicken peripheral blood lymphocytes,which was inserted in-to 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%),andβ-turn(5.70%).IRF7 amino acid of chicken had low homology with other avian species,human and mammals,and the five functional do-mains were not conserved among them.The subcellular localization of fusion protein EGFP-chIRF7 changed from the cy-toplasm to the nucleus in DF-1 cells treated with polyinosinic-polycytidylic acid[Poly(I∶C)]or NDV.Meanwhile,fusion protein EGFP-chIRF7 overexpression reduced the replication ability and cytopathogenicity of NDV.chIRF7 gene was ex-pressed 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 pecto-ral 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 develop-mental stages,indicating the potential regulatory role in the development of these three tissues.