松江鲈IL-15 Rα的结构特征与表达分析  

Characterization and expression analysis of interleukin-15Rαfrom Trachidermus fasciatus

作　　者：林日锦 隗涵涵 钟金妙 刘莹莹 LIN Rijin;WEI Hanhan;ZHONG Jinmiao;LIU Yingying(School of Marine Science, Shandong University (Weihai), Weihai Shandong 264209, China)

机构地区：[1]山东大学(威海)海洋学院,山东威海264209

出　　处：《海洋渔业》2020年第4期394-402,共9页Marine Fisheries

基　　金：山东省自然科学基金项目(ZR2016CP10);威海市科技局项目(1070413421706)。

摘　　要：白介素15受体α链(IL-15 Rα)是介导IL-15信号转导的高亲和力膜受体复合物的重要亚基。通过RACE技术克隆得到松江鲈(Trachidermus fasciatus Heckel IL-15 Rα)基因cDNA全长序列(命名为TfIL-15Rα),TfIL-15Rα全长为998 bp,包括5′-非编码区(5′-UTR)85 bp,开放阅读框(ORF)663 bp和3′UTR 250 bp。基因ORF编码220个氨基酸(aa),前27 aa为信号肽序列。同源比对发现,松江鲈TfIL-15Rα与其他鱼类的序列一致性为19%~46%,保守度较低。多序列比对结果显示,IL-15Rα保守序列主要分布于sushi结构域(31~95 aa),4个高度保守的半胱氨酸形成的两对二硫键,是结构域发挥配体结合功能的重要位点。qRT-PCR分析表明,TfIL-15Rα广泛表达于松江鲈各组织中。腹腔注射LPS后,在皮肤、血液和肝脏中,刺激后2 h,IL-15Rα表达量迅速上调至最高峰,分别为对照组的14倍、52倍和21倍;而在脾脏中,刺激后12 h,TfIL-15RαmRNA达到最大表达量。由以上实验结果推测,TfIL-15Rα参与到了松江鲈抵抗外界刺激的先天免疫过程中。IL-15 receptor complex consists of three subunits,including IL-Rα,IL-Rβ,and IL-Rγc.IL-2 and IL-15 share IL-Rβand IL-Rγc as parts of common receptors while IL-Rαacts as a high-affinity,specific subunit for IL-15 binding with IL-15R.To reveal its function in the innate immune system,the IL-15 RαcDNA(named as TfIL-15Rα)was cloned from roughskin sculpin Trachidermus fasciatus via the rapid amplification of cDNA ends(RACE)approaches.The full-length of IL-15 RαcDNA was 998 bp,including a 5′untranslated region(5′-UTR)85 bp,an open reading frame(ORF)663 bp,and a 3′untranslated region(3′UTR)250 bp.The ORF encoded 220 amino acids(aa)with a 27aa signal peptide sequence.The mature peptide had a total length of 193aa,with a predicted molecular weight of 21.042 kDa,and a theoretical isoelectric point of 6.36.The protein sequence shared 19%-46%identity with reported fish IL-15 Rα,displaying relatively low conservation.Multiple alignments of IL-15 Rαshowed that the region with high identity was mainly located in the sushi domain(31aa-95aa)which was crucial for cytokine IL-15 recognition.The two disulfide bonds in the sushi domain formed with four conserved cysteines were important sites for ligand binding.Quantitative real-time PCR(qPCR)was used to determine the distribution of TfIL-15Rαexpression in 10 tissues(blood,heart,liver,gill,intestine,skin,kidney,spleen,brain,egg)and the temporal expression profiles at 2 h,6 h,12 h,24 h,48 h,72 h,96 h post LPS(lipopolysaccharide)challenge in the skin,blood,liver,and spleen.The results showed that TfIL-15Rαwas widely expressed in different tissues of healthy fish,with the highest expression in the gill,intestine and egg.For post LPS challenge,TfIL-15Rαincreased rapidly to the maximum of 14 times,52 times and 21 times compared with that of the control group at 2 h post challenge(hpc)in the skin,blood and liver.The TfIL-15RαmRNA was induced to the maximum at 12 h post challenge in the spleen.The relatively time lag in response post immune activation may be due to tha

关 键 词：松江鲈 白介素15受体α链(IL-15Rα) 克隆 重组蛋白表达 

分 类 号：S917[农业科学—水产科学]