Sequence Characteristics of Interleukin-1 Receptor-Related Kinase 3 (IRAK-3) in Ayu (Plecoglossus altivelis) and Its Immune Correlation Analysis Against Vibrio anguillarum Infection
LI Shang-Yang1,2, ZHOU Yan1,2, CHAI Fang-Chao2, ZHOU Qian-Jin1,2,*, CHEN Jiong1,2
1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China; 2 School of Marine Science, Ningbo University, Ningbo 315832, China
Abstract：Interleukin-1 receptor-associated kinase-3 (IRAK-3) is a member of the IRAK family. It can regulate immune homeostasis and tolerance in a number of infectious and non-infectious diseases through its inhibition of pro-inflammatory cytokine production. Recently, few studies are focusing on the function of IRAK-3 in fish. Herein, an IRAK-3 transcript (PaIRAK-3) was obtained from the monocyte/macrophage transcriptome of ayu (Plecoglossus altivelis). It possessed an open reading frame of 1 722 bp, predictedly encoding 573 amino acids, and had a theoretical molecular weight of 63.2 kD. PaIRAK-3 exhibited a N-terminal death domain, a proline/serine/threonine-rich domain, a kinase and/or pseudokinase domain and a C-terminal domain of IRAK family. The phylogenetic tree showed that IRAK-3 was relatively conservative in fish, and PaIRAK-3 had the highest homology with rainbow trout (Oncorhynchus mykiss) (63.1%) and silver salmon (Oncorhynchus kisutch) (63.0%). Quantitative PCR analysis showed that PaIRAK-3 was mainly expressed in the kidney, gill, spleen, liver and intestine. After Vibrio anguillarum infection, PaIRAK-3 was significantly up-regulated at the mRNA level in the kidney, spleen and liver of ayu, reaching the maximum value at 8 h post infection. And the mRNA level of PaIRAK-3 increased only at 8 h post infection in the gill tissue. Recombinant PaIRAK-3 (rPaIRAK-3) was prokaryotically expressed using BL21 Rosetta strain of Escherichia coli, and the purified rPaIRAK-3 was used to immunize mice (Mus musculus) to prepare the polyclonal antibodies. Western blot analysis using the polyclonal antibodies against rPaIRAK-3 showed that PaIRAK-3 was significantly up-regulated against V. anguillarum infection at the protein level in the liver and spleen tissue and reached the maximum value at 12 h post infection. In conclusion, PaIRAK-3 is closely related to V. anguillarum infection, which provides a premise for further research on the antibacterial immunity of PaIRAK-3.
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