Molecular cloning, expression and functional characterization of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) in grass carp, Ctenopharyngodon idella
Molecular cloning, expression and functional characterization of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) in grass carp, Ctenopharyngodon idella
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草鱼肿瘤坏死因子（TNF）受体相关因子（TRAF）相互作用蛋白（TRIP）的分子克隆、表达和功能表征
DOI：
10.1016/j.fsi.2016.08.041
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发表时间：
2016
影响因子：
4.7
通讯作者：
Ji H.
中科院分区：
文献类型：
--
作者：
Lu R. -H.;Chang Z. -G.;Sun J.;Yang F.;Nie G. -X.;Ji H.
TRIP(肿瘤坏死因子受体相关因子(TRAF)相互作用蛋白)是肿瘤坏死因子超家族的成员，在脊椎动物的炎症过程中起着重要的调节作用。然而，硬骨鱼中没有关于Trip的信息。本研究克隆了草鱼TRIP基因的全长，包括112个碱基的5‘非编码区，1359个碱基的开放阅读框和29个碱基的3’非编码区。该基因编码一个452个氨基酸的蛋白质，估计分子质量为51.06 kD，预测的理论等电点(PI)为9.11。实时荧光定量聚合酶链式反应分析表明，TRIPmRNA在草鱼各组织中均有表达，其中以肾脏表达最高，其次是肠道和胸腺。然而，在脂肪、脾、肝脏、性腺和心脏中也检测到较低水平的表达。亚细胞定位和双杂交分析表明，TRIP定位于细胞核内，并与HEK293T细胞中的TRAF1和TRAF2相互作用。此外，与肿瘤坏死因子-α类似，IL-10和TRIPmRNA在高脂肪或高碳水化合物饲料喂养的鱼类的脾中表达上调，提示TRIP可能与对过量能量摄入的反应有关。2μg/mL脂多糖作用4h后，IDELLA肝细胞TRIPmRNA相对表达水平显著降低(P<0.05)，而炎性细胞因子TRAN-α和IL-10表达水平显著升高(P<；P<0.05)。综上所述，这些结果表明TRIP可能在草鱼的免疫防御中发挥重要作用，并有可能成为草鱼抗炎的靶点。
TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5′UTR of 112 bp, an ORF of 1359 bp, and a 3′UTR of 29 bp before the poly (A) tail, was cloned from grass carp,Ctenopharyngodon idella. TheTRIPgene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed thatTRIPmRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 andTRIPmRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P< 0.05) after hepatocyte ofC.idellawas treated with 2 μg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P< 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp.