尼罗罗非鱼干扰素调节因子1的表达与细胞定位

为了分析尼罗罗非鱼干扰素调节因子1(IRF1)的表达规律及其功能,克隆了尼罗罗非鱼IRF1基因c DNA全长,与其他脊椎动物IRF1基因进行比对,构建IRF家族系统进化树。然后使用Poly I∶C对尼罗罗非鱼进行体内注射诱导抗病毒免疫反应,利用荧光定量PCR技术检测处理组和对照组中尼罗罗非鱼各组织IRF1基因的表达差异。最后构建尼罗罗非鱼IRF1基因真核表达载体,对其进行细胞定位。结果表明,尼罗罗非鱼IRF1有888 bp的开放读码框,编码295个氨基酸的蛋白序列。该蛋白N端高度保守,并含有6个保守的色氨酸,具有DNA结合结构域。对蛋白相似率的计算发现尼罗罗非鱼与斑马鱼IRF1相似度为52.1%。而与大鼠IRF1的相似率最低,为36.2%。IRF家族成员蛋白序列构建的系统进化树显示,IRF家族共分为4个亚群。将p CMV3-FLAG-IRF1真核表达载体转染到Hela细胞中后,对IRF1进行细胞定位发现,尼罗罗非鱼IRF1主要分布在胞质中,胞核中有少量分布。对尼罗罗非鱼使用Poly I∶C进行体内注射诱导抗病毒应答反应,利用实时荧光定量PCR检测到各个组织中IRF1在不同时间下的表达水平有显著差异。本研究完成了尼罗罗非鱼IRF1的表达和细胞定位,对各种脊椎动物IRF家族成员进行了系统进化分析,结果有助于深入了解干扰素系统在机体免疫系统中的调控机制。

Expression Characterization of Interferon Regulatory Factor 1 in Nile Tilapia

In order to study the expression and function of interferon Regulatory Factor (IRF1)in nile tilapia, the full length cDNA of IRF1 was cloned and applied for phylogenetic analysis,and the expression variation was checked by qRT-PCR after PolyI∶C stimulation.Cell transfection by eukaryotic expression vector was used to analy-zing for subcellular localization.The opening reading frame of nile tilapia IRF1 contains 888 bp with deduced pro-tein of 295 amino acids.Structurally,nile tilapia IRF1 protein typically shares the conserved characterizations with other species′IRF1 homologues,displaying conserved DNA-binding domain,IRF association domain,serine-rich C terminal domain,and 6 tryptophan residues in the N terminal.The results of phylogenetic analysis showed that the IRF family was divided into four subfamily:IRF1 ,IRF3,IRF4 and IRF5.The nile tilapia IRF1 was found to be mer-ged into the teleost subgroup,amphibian and mammalian homologues were clustered into their corresponding sub-groups.Real-time PCR revealed a broad expression pattern of nile tilapia IRF1 in various tissues.The expression of IRF1 was mainly in the head kidney,spleen,liver and gill,furthermore,which increased obviously after PolyI ∶C stimulation,especially for the spleen and head kidney.Subcellular localization analysis showed that nile tilapia IRF1 mainly resides in the cytoplasm.These data supported the view that nile tilapia IRF1 was a potential molecule in IFN immune defense system against viral infection.