日本鳗鲡TLR21基因的鉴定、免疫应答与启动子分析

摘要

为研究TLR21(Toll like receptor 21)在低等脊椎动物中的功能及表达调控机制,我们扩增获得了日本鳗鲡TLR21(AjTLR21)cDNA序列,其编码的蛋白具有TLR家族的共同特征.AjTLR21基因结构与其他鱼类和两栖类TLR21相同,由单个外显子编码.荧光定量结果显示,AjTLR21在血液、鳃、脾脏、中肾等11个组织/器官中转录表达,其中在血液中表达量最高.经Poly I:C诱导后8h,AjTLR21在脾脏和中肾中的表达量显著性上调;诱导后16h,AjTLR21在血液、鳃、肠和脾脏中的表达量显著性上调(P<0.05).双荧光素酶报告基因结果显示,在AjTLR215′上游调控序列-1179 bp到+117 bp存在Poly I:C调节的正调控元件.经Edwardsiella tarda诱导后16h和72h,AjTLR21分别在血液和中肾组织的表达量显著性上调,表明AjTLR21同时也参与了抗细菌免疫应答,其在机体免疫系统中的功能具有多样性.研究对于理解日本鳗鲡AjTLR21的免疫学功能具有重要的理论意义和应用价值.%Toll like receptor 21 (TLR21) is a non-mammalian Toll-like receptor that recognizes unmethylated CpG DNA to trigger innate immune responses against pathogenic microorganism infection. The present study cloned the full-length cDNA sequence of TLR21 (AjTLR21) from Japanese eel, Anguilla japonica, which had the common charac-teristics of Toll like receptors (TLRs). The intronless AjTLR21 shared similar gene structure with other vertebrate TLR21 genes. AjTLR21 widely expressed in all examined tissues/organs with the highest expression in blood. Poly I:C treatment significantly induced the expression of AjTLR21 in spleen and kidney at 8 hours and in blood, gill, intestine and spleen at 16 hours (P<0.05). Luciferase reporter assay exhibited that the 5′ flanking region of the AjTLR21 contains positive regulatory elements for Poly I:C induced AjTLR21 expression. The stimulation of Edwardsiella tarda also in-creased the expression of AjTLR21 in blood and kidney at both 16h and 72h, suggesting an important role in anti-bac-terium immune response. Our results revealed multiple effects of teleosts TLR21 in immune system, which has impor-tant theoretical significance and application value to understand the immunological function of AjTLR21.