尼罗罗非鱼白细胞介素21基因(OnIL-21)及其受体基因(OnIL-21R)克隆及表达分析  

Identification and expression analysis of interleukin 21(IL-21) and its receptor(IL-21R) in Nile tilapia(Oreochromatidus niloticus)

作　　者：李兰[1] 高阿龙 陈建林 雷阳 吴丽婷 叶剑敏[1] LI Lan;GAO Along;CHEN Jianlin;LEI Yang;WU Liting;YE Jianmin(Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture,Institute of Modern Aquaculture Science and Engineering,School of Life Sciences,South China Normal University,Guangzhou 510631,China)

机构地区：[1]华南师范大学生命科学学院,现代水产养殖科学与工程研究院,广东省水产健康安全养殖重点实验室,广东广州510631

出　　处：《水产学报》2022年第11期2038-2052,共15页Journal of Fisheries of China

基　　金：国家自然科学基金(31972818);中国博士后科学基金(2019M662959)。

摘　　要：为研究白细胞介素21 (IL-21)及其受体IL-21R在鱼类抵御病原菌的免疫应答过程中的作用及在机体炎症中的分子机制,实验利用反转录PCR (RT-PCR)技术成功克隆了尼罗罗非鱼IL-21基因(OnIL-21)及其受体IL-21R基因(OnIL-21R),对其进行生物信息学分析,并利用实时荧光定量PCR (qRT-PCR)技术检测其mRNA的表达水平。结果显示,OnIL-21及OnIL-21R的开放阅读框分别为420 bp和1 548 bp,分别编码139和515个氨基酸。经氨基酸序列预测分析,OnIL-21为分泌型蛋白,具有多个保守的磷酸化位点和一个Ig-like结构域;而OnIL-21R是跨膜蛋白,具有典型的γ-c链,氨基酸序列都具有多个保守的磷酸化位点和糖基化位点。系统进化树结果表明,IL-21与IL-21R的氨基酸序列在物种进化过程中具有一定的保守性,尼罗罗非鱼IL-21和IL-21R的氨基酸序列均与斑马拟丽鱼IL-21和IL-21R的亲缘关系最近。组织差异性表达分析发现,OnIL-21及OnIL-21R在健康尼罗罗非鱼的多种组织中均有表达,但具有明显的组织差异性。OnIL-21在皮肤和心脏组织中表达量较高,在肌肉中较低;而OnIL-21R在鳃和脾脏组织中表达量最高,在肌肉中最低。无乳链球菌和嗜水气单胞菌体内感染和体外刺激白细胞后,头肾和脾脏中OnIL-21和OnIL-21R表达量均呈现显著上调,并在72 h内维持较高的表达水平,说明OnIL-21和OnIL-21R可能参与尼罗罗非鱼病原菌感染的免疫应答过程。此外,重组蛋白(r)OnIL-21刺激脾脏白细胞后,OnIL-21R和炎症相关因子基因(IL-1β、TNF-α、IFN-γ、IL-6和IL-10)的表达均发生显著上调,表明OnIL-21可调控其受体基因OnIL-21R的表达及参与调控机体的炎症反应。上述研究结果阐述了尼罗罗非鱼IL-21与IL-21R的分子生物学特征,并初步阐明了二者参与机体病原菌感染的免疫应答过程,为进一步探究IL-21通过其受体IL-21R调控机体免疫反应的作用机制和信号通路提供了重要参考。Interleukin 21(IL-21) is a kind of pleiotropic cytokine, mainly through its specific receptor(IL-21R) to exercise immunomodulatory function, including promoting T cells proliferation, regulating B cells differentiation and enhance the cytotoxicity of natural killer cells(NK), and play an important role in the immune response to pathogen infection. In order to study the role of IL-21 and its receptor IL-21R in the immune response of fish against pathogenic bacteria, homologs of IL-21 and IL-21R in Oreochromis niloticus were identified, and their roles in bacterial infection and the regulation of inflammatory response were investigated. The open reading frame of OnIL-21 and OnIL-21R are 420 bp and 1 548 bp, encoding 139 and 515 amino acids, respectively. The deduced OnIL-21 is a secreted protein containing an Ig-like domain. OnIL-21R is a transmembrane protein, contains two conserved cysteine residues, a FNⅢ domain and a highly conserved WSXWS motif of the type I cytokine receptor family. Both OnIL-21 and OnIL-21R contained multiple conserved phosphorylation sites and glycosylation sites.Expression analysis indicate that the OnIL-21 exhibited constitutive expression in the examined tissues, with the highest expression in skin. OnIL-21R was also widely expressed in multiple tissues, with the highest expression in gill and spleen. In addition, the OnIL-21 and OnIL-21R expressions are significantly up-regulated in spleen and anterior kidney following challenges of Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro,and maintained high expression level within 72 h, which suggested that OnIL-21 and OnIL-21R may get involved in host defense against bacterial infection. Further, after being stimulated with recombinant protein(r)OnIL-21 in vitro, the expressions of OnIL-21R and inflammation-related cytokines such as interleukin-1β(IL-1β), tumor necrosis factor α(TNF-α), interferon γ(IFN-γ), interleukin 6(IL-6) and interleukin 10(IL-10) were up-regulated significantly in spleen lymphocytes, which

关 键 词：尼罗罗非鱼 IL-21 IL-21R 基因克隆 免疫应答 

分 类 号：S942[农业科学—水产养殖]