To study the interrelationship between the signaling adaptors of innate pattern recognition receptor (PRR) pathways including toll-like receptor (TLR), retinoic acid-inducible gene-1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and cytoplasmic DNA recognition receptors (CDR) pathways. The coding genes of porcine TRIF, MAVS, STING, MyD88, RIPK2, and ASC were isolated from PK15 cells. Phylogenetic analysis of the six adaptor proteins in pig, cattle, goat, horse, human, mouse, chicken, and duck performed by MEGA 5.05 showed that these adaptors have slightly different similarity across species. The expression of these proteins in transfected cells were detected by both Western blotting and confocal microscopy. All six adaptors were visualized in cytoplasm but with different distribution patterns. The activities of the six adaptors triggering NF-κB and ISRE signaling and downstream gene productions were examined by dual-luciferase reporter assay and real-time RT-PCR, respectively. The results showed that STING has an ability to activate ISRE signaling, MyD88, RIPK2 and ASC possess NF-κB signal activity, while TRIF and MAVS can activate both. Furthermore, the mutual signaling effects were assessed by NF-κB and ISRE dual-luciferase reporter assay in the co-expression experiments. STING was shown to enhance MAVS activated NF-κB signaling and MyD88 could heighten STING activated ISRE signaling. However, all other adaptors inhibited each other to varying degrees. The work provides a global insight of porcine innate immune signaling pathways and their interaction network.

中文翻译：

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猪先天免疫途径的信号衔接子的相互关系分析。

研究先天模式识别受体（PRR）信号通路的信号衔接子之间的相互关系，包括通行费样受体（TLR），视黄酸诱导基因-1样受体（RLR），核苷酸结合寡聚化域样受体（NLR） ）和胞质DNA识别受体（CDR）途径。从PK15细胞中分离出猪TRIF，MAVS，STING，MyD88，RIPK2和ASC的编码基因。通过MEGA 5.05对猪，牛，山羊，马，人，小鼠，鸡和鸭中的六个衔接子蛋白进行的系统发育分析表明，这些衔接子在物种间的相似性略有不同。通过蛋白质印迹和共聚焦显微镜检测转染细胞中这些蛋白质的表达。所有六个衔接子均在细胞质中可见，但分布模式不同。分别通过双荧光素酶报告基因测定和实时RT-PCR检查了六个触发NF-κB和ISRE信号转导的衔接子的活性以及下游基因的产生。结果表明，STING具有激活ISRE信号的能力，MyD88，RIPK2和ASC具有NF-κB信号活性，而TRIF和MAVS均可激活。此外，在共表达实验中，通过NF-κB和ISRE双荧光素酶报告基因分析评估了相互的信号传导作用。STING被证明可以增强MAVS激活的NF-κB信号传导，而MyD88可以增强STING激活的ISRE信号传导。但是，所有其他适配器在不同程度上互相抑制。这项工作提供了猪先天免疫信号通路及其相互作用网络的全球见解。