GPATCH3, a protein with G-patch domain, is known to participate in innate immune response and organ development in mammals. However, there are few reports on GPATCH3 in fish. Here the cDNA sequence of GPATCH3 was cloned from Ctenopharyngodon idella (CiGPATCH3, MN149902) and was determined its character. A cDNA sequence of CiGPATCH3 is 1646 bp and contains an ORF of 1221 bp translating a protein of 407 amino acids. Phylogenetic analysis uncovered that CiGPATCH3 possesses a relatively high degree of homology with Cyprinus carpio GPATCH3. The mRNA level of CiGPATCH3 was increased following the intracellular stimulation of poly (I:C) into CIK cells. In vivo, over-expression of CiGPATCH3 can significantly up-regulate IFN 1 and ISG15 expression at mRNA and protein levels. To investigate the molecular mechanism by which GPATCH3 initiates the innate immune response in fish, co-IP experiments were performed to analyze the substrates of CiGPATCH3. The results showed that CiGPATCH3 directly interacted with CiSTING, but not with CiIRF3, CiIRF7, CiTBK1 or CiIPS-1. As compared with the single transfection of CO cells with either CiGPATCH3 or CiSTING, the expression of IFN 1 was more significantly up-regulated in cells under treatment with dual transfection of CiGPATCH3 and CiSTING. Knockdown of CiGPATCH3 inhibited STING-mediated IFN 1 expression in fish cells. Over-expression of CiGPATCH3 and CiSTING facilitated the phosphorylation and cytoplasmic-to-nuclear translocation of CiIRF7. These results explicitly showed that CiGPATCH3 up-regulates IFN 1 and ISG15 expression via the activation of STING-IRF7 signal axis in vivo.

GPATCH3 是一种具有 G-patch 结构域的蛋白质，已知参与哺乳动物的先天免疫反应和器官发育。然而，关于鱼类中 GPATCH3 的报道很少。在这里，GPATCH3的 cDNA 序列是从Ctenopharyngodon idella ( CiGPATCH3 , MN149902) 中克隆出来的，并确定了它的特性。CiGPATCH3的 cDNA 序列为 1646 bp，包含 1221 bp 的 ORF，翻译 407 个氨基酸的蛋白质。系统发育分析发现Ci GPATCH3与Cyprinus carpio GPATCH3具有较高的同源性。在细胞内刺激 poly (I:C) 进入 CIK 细胞后，CiGPATCH3的 mRNA 水平增加。在体内，Ci GPATCH3 的过表达可以在 mRNA 和蛋白质水平上显着上调 IFN 1 和 ISG15 的表达。为了研究 GPATCH3 启动鱼先天免疫反应的分子机制，进行了 co-IP 实验来分析Ci GPATCH3的底物。结果表明，Ci GPATCH3 直接与Ci STING相互作用，但不与Ci IRF3、Ci IRF7、Ci TBK1 或Ci IPS-1相互作用。与用Ci GPATCH3 或Ci单次转染 CO 细胞相比STING，在Ci GPATCH3和Ci STING双重转染处理的细胞中，IFN 1的表达更显着上调。Ci GPATCH3 的敲低抑制了鱼细胞中 STING 介导的 IFN 1 表达。过表达的次GPATCH3和次STING促进的磷酸化和细胞质到核易位次IRF7。这些结果明确表明，Ci GPATCH3 通过体内STING-IRF7 信号轴的激活上调 IFN 1 和 ISG15 的表达。