赤眼鳟Toll样受体3基因cDNA全长克隆及表达分析

为探究赤眼鳟（Squaliobarbus curriculus）Toll样受体3（Toll-like receptor 3，ScTLR3）基因是否参与抗病毒免疫反应，实验运用RACE技术，克隆得到ScTLR3基因cDNA全长序列，并进行了生物信息学分析；通过Real-Time qPCR技术，检测了ScTLR3 mRNA在健康赤眼鳟10个组织中的分布以及感染草鱼呼肠孤病毒（GCRV）后肝脏、脾脏、体肾和头肾中的表达特征。结果表明:ScTLR3基因cDNA序列全长4043 bp，包括5-非编码区（UTR）216 bp，开放阅读框（ORF）2715 bp和3-UTR 1112 bp；ScTLR3共编码904个氨基酸残基，推导的蛋白分子量102.67 kD，理论等电点8.76；SMART结构域预测显示，ScTLR3由N端的信号肽（SP）、富亮氨酸结构域（LRRs）、跨膜结构域（TM）和C端的Toll/白介素-1受体结构域（TIR）组成。实时荧光定量结果显示，ScTLR3mRNA在检测的各组织中均有表达，肝脏中的相对表达量极显著高于其他组织（P0.01）；感染GCRV后，肝脏、脾脏、体肾和头肾组织中ScTLR3 mRNA均上调表达，肝脏、脾脏和体肾组织中的相对表达量在24h达到峰值，分别为对照组的5倍、7倍和6倍。研究表明，ScTLR3具有TLRs家族基因的典型结构特征，并能被GCRV诱导表达，推测其在赤眼鳟抗GCRV入侵免疫反应中发挥了重要作用。

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF TOLL-LIKE RECEPTOR 3 GENE IN BARBEL CHUB SQUALIOBARBUS CURRICULUS

To investigate whether the toll-like receptor 3 (ScTLR3) gene involve in antiviral immune response, TLR3 full-length cDNA in barbel chub (Squaliobarbus curriculus) was cloned and characterized with RACE technique and grass carp reovirus (GCRV) infection was used The method of real-time qPCR was used to examine the expression of ScTLR mRNA in 10 different tissues and the relative expression in liver, spleen, trunk kidney and, head kidney after infection at different times. The results showed that the full-length cDNA of ScTLR3 was 4043 bp including a 216 bp 5'-terminal untranslated region (UTR), an 1112 bp 3'-terminal untranslated region (UTR) and a 2715 bp open reading frame (ORF) encoding a polypeptide of 904 amino acid residues. The putative isoelectric point (pI) and molecular weight (Mw) of ScTLR3 were 8.76 and 102.67 kD, respectively. The ScTLR motifs were consisted of N-terminal signal peptide (SP), 14 leucine-rich repeats (LRRs), transmembrane domain (TM), and the Toll/IL-1 Receptors domain (TIR) in C-terminal. ScTLR3 mRNA was expressed in all tested tissues, and its expression in liver was extremely significant higher than other tested tissues in health barbell chub (P0.01). GCRV infection induced the ScTLR expression in liver, spleen, trunk kidney, and head kidney. Compared with the control group, the ScTLR mRNA expression levels in liver, spleen and trunk kidney tissues of barbel chub was induced 5-, 7-, and 6-fold at 24h post infection, respectively. These results suggest that ScTLR3 may play an important role in the immune response of barbel chub against GCRV infection.