【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。更多还原

【Abstract】 IL-15 receptor complex consists of three subunits, including IL-Rα, IL-Rβ, and IL-Rγc. IL-2 and IL-15 share IL-Rβ and IL-Rγc as parts of common receptors while IL-Rα acts as a high-affinity, specific subunit for IL-15 binding with IL-15 R. To reveal its function in the innate immune system, the IL-15 Rα cDNA(named as TfIL-15 Rα) was cloned from roughskin sculpin Trachidermus fasciatus via the rapid amplification of cDNA ends(RACE) approaches. The full-length of IL-15 Rα cDNA 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 27 aa signal peptide sequence. The mature peptide had a total length of 193 aa, 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 Rα showed that the region with high identity was mainly located in the sushi domain(31 aa-95 aa) 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-15 Rα expression 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-15 Rα was widely expressed in different tissues of healthy fish, with the highest expression in the gill, intestine and egg. For post LPS challenge, TfIL-15 Rα increased 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-15 Rα mRNA 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 that the spleen is considered to be the most important hematopoietic organ for production of lymphocytes, monocytes, and reticuloendothelial cells in teleost fish rather than a direct immune response tissue compared with the skin, blood and liver. Worth noting, TfIL-15 Rα increased again with a 25-fold induction at 96 hpc in the liver. This increase could be attributed to the upregulation in additional cell types activated by signaling molecules from the initial immune response. These results suggest that TfIL-15 Rα may play important roles in fish innate immune response against microbial infections.更多还原