【摘要】 【目的】明确吉富罗非鱼干扰素a3(IFNa3)基因的结构特征及组织表达模式，以及聚肌胞苷酸[Poly(I:C)]刺激和罗非鱼湖病毒（TiLV）感染后的表达变化，并探究IFNa3基因对干扰素刺激基因（ISGs）及TiLV复制的影响，为科学制定罗非鱼养殖疾病控制策略提供理论依据。【方法】克隆吉富罗非鱼IFNa3基因及进行生物信息学分析，采用实时荧光定量PCR检测IFNa3基因在健康吉富罗非鱼组织中的表达分布及Poly(I:C)刺激和TiLV感染后的表达变化，并通过过表达和敲降基因试验检测吉富罗非鱼IFNa3基因对ISGs表达及TiLV复制的影响。【结果】吉富罗非鱼IFNa3基因开放阅读框（ORF）长度为570 bp，共编码189个氨基酸残基。吉富罗非鱼IFNa3蛋白含有1个IFabd结构域，具有Ⅰ型IFN典型的结构特征。吉富罗非鱼IFNa3氨基酸序列与其他硬骨鱼类Ⅰ型IFNa3氨基酸序列的相似性为70.3%～83.5%，其中与伯氏妊丽鱼IFNa3氨基酸序列的相似性最高（83.5%）。IFNa3基因在健康吉富罗非鱼的脾脏、肝脏、脑、鳃、肠道、肌肉、心脏及头肾等组织中均有表达，以鳃组织中的相对表达量最高；经Poly(I:C)刺激和TiLV感染后，吉富罗非鱼IFNa3基因在鳃、头肾和脾脏等组织中的表达均呈上调表达，且呈现“早期升高、随后回落”的时序变化特征。在罗非鱼脑细胞（TiB）中过表达IFNa3基因，能极显著上调STAT1、ISG15和Mx基因的表达（P<0.01，下同），而TiLV-S10基因相对表达量极显著降低；通过siRNA敲降IFNa3基因表达后，TiLV-S10基因相对表达量极显著上升，而STAT1、ISG15和Mx基因的表达极显著下调。【结论】吉富罗非鱼IFNa3具有鱼类Ⅰ型IFN典型的结构特征，受免疫刺激后在鳃、头肾和脾脏等组织中均呈上调表达，通过激活JAK-STAT信号通路并诱导下游多种ISGs表达，在细胞水平上抑制TiLV复制，进而在吉富罗非鱼抗TiLV感染的先天免疫应答中发挥关键作用。更多还原

【Abstract】 【Objective】This study aimed to investigate the structural characteristics and tissue expression pattern of the interferon a3(IFNa3) gene in GIFT Oreochromis niloticus, to examine its expression change after polyinosinic acid [Poly(I:C)] stimulation and tilapia lake virus(TiLV) infection, and to discover effects of IFNa3 gene on interferonstimulated genes(ISGs) and TiLV replication, providing a theoretical basis for disease prevention and control strategies in tilapia aquaculture. 【Method】The IFNa3 gene in GIFT Oreochromis niloticus was cloned and subjected to bioinformatic analysis, and real-time fluorescence quantitative PCR was employed to examine expression distribution of IFNa3 gene in healthy tissues of GIFT Oreochromis niloticus and expression change after Poly(I:C) stimulation and TiLV infection. Effects of IFNa3 of GIFT Oreochromis niloticus on ISG expression and TiLV replication were evaluated by gene experiments of overexpression and knockdown. 【Result】The open reading frame(ORF) of IFNa3 gene of GIFT Oreochromis niloticus was 570 bp, encoding 189 amino acid residues. The IFNa3 protein contained one IFabd domain, showing typical structure characteristic of typeⅠIFN. The similarity between IFNa3 amino acid sequence of GIFT Oreochromis niloticus and typeⅠIFNa3 amino acid sequence in other teleost fish species was 70.3%-83.5%, with the highest similarity observed with the IFNa3 amino acid sequence of Haplochromis burtoni(83.5%). The IFNa3 gene was expressed in tissues of spleen, liver, brain, gill, intestine, muscle, heart, and head kidney, with the highest expression in gill tissues. After Poly(I:C) stimulation and TiLV infection, IFNa3 expression was up-regulated in the gill, head kidney, and spleen, displaying a temporal pattern of initial increasing and subsequent decreasing. The overexpressed gene IFNa3 of tilapia brain(TiB) cells could extremely up-regulate the expression of STAT1, ISG15, and Mx(P<0.01, the same below), while extremely down-regulate the relative expression of TiLV-S10 gene. After knocking down the expression of IFNa3 gene by siRNA, the relative expression of Ti LV-S10 was extremely significantly increased, but the expressions of STAT1, ISG15, and Mx genes were extremely significantly decreased. 【Conclusion】The IFNa3 of GIFT Oreochromis niloticus exhibits typical structure characteristics of typeⅠIFN, and is up-regulated in tissues of gill, head kidney, and spleen after immune stimulation. It activates JAK-STAT signaling pathway, and induces multiple downstream expressions of ISGs, and suppresses TiLV replication at the cellular level, thereby playing a critical role in the innate immune defense against TiLV infection in GIFT Oreochromis niloticus.更多还原