| 摘要: |
| CC趋化因子受体3（CCR3）是CC型趋化因子的受体，属于G蛋白耦联受体（GPCRs）家族成员，与过敏等多种炎性疾病密切相关。为探讨香鱼CCR3（PaCCR3）在响应鳗弧菌感染时的表达变化，从转录组测序数据中获得了PaCCR3基因全长cDNA序列。序列分析表明，PaCCR3具有7次跨膜结构，与胡瓜鱼CCR3氨基酸序列同源性最高（84.6%）。系统进化树分析表明，哺乳类、两栖类和鱼类CCR3单独成簇，鱼类CCR3形成一个大簇；PaCCR3与胡瓜鱼CCR3进化相关性最高。实时荧光定量PCR结果显示，PaCCR3基因mRNA在单核/巨噬细胞（MO/MФ）中表达量最高；鳗弧菌感染后肝、脾和头肾中PaCCR3基因mRNA表达量显著上调；且香鱼MO/MФ经鳗弧菌体外刺激后，PaCCR3基因mRNA的表达量显著上调。Western blot分析表明经鳗弧菌体外刺激后，香鱼MO/MФ中PaCCR3蛋白的表达量也显著增加。综上，PaCCR3基因mRNA及其编码蛋白响应于病原体感染而被诱导表达。研究结果首次揭示了香鱼CCR3基因mRNA及蛋白的表达响应于鳗弧菌侵染，它可能通过介导MO/MФ的功能活性参与免疫应答，可为深入探究鱼类CCR3的功能及作用机制提供新的切入点。 |
| 关键词: 香鱼(Plecoglossus altivelis) CCR3 单核/巨噬细胞 鳗弧菌 表达分析 |
| DOI：10.11693/hyhz20210500118 |
| 分类号:Q786;S917 |
| 基金项目:国家自然科学基金项目,31972821号;浙江省自然科学基金项目,LY21C190003号;宁波大学研究生科研创新基金项目,IF2020138号;农产品质量安全危害因子与风险防控国家重点实验室自主设计课题,2010DS700124-ZZ2008号。 |
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| MOLECULAR IDENTIFICATION OF CC MOTIF CHEMOKINE RECEPTOR 3 (CCR3) GENE AND ITS RESPONSE TO VIBRIO ANGUILLARUM INFECTION IN AYU PLECOGLOSSUS ALTIVELIS |
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ZHANG Ling-Ling1,2, YU Li2, LI Chang-Hong1,2, MIAO Liang1,2, CHEN Jiong1,2
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1.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts, Ningbo University, Ningbo 315211, China;2.Laboratory of Biochemistry and Molecular Biology, School of Marine Science, Ningbo University, Ningbo 315832, China
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| Abstract: |
| The CC chemokine receptor 3 (CCR3), a kind of CC motif chemokine receptor, belongs to the family of G protein-coupled receptors (GPCRs). CCR3 is closely associated with various inflammatory diseases such as allergies. In order to explore the expression changes of ayu (Plecoglossus altivelis) CCR3 (PaCCR3) in response to Vibrio anguillarum infection, the full-length cDNA sequence of PaCCR3 gene was obtained from ayu monocytes/macrophages (MO/MФ) transcriptome sequencing data. Sequence analysis showed that PaCCR3 had a seven-pass transmembrane domain, and had the highest amino acid sequence homology with the CCR3 of rainbow smelt (Osmerus mordax) (84.6%). Phylogenetic tree analysis showed that mammals, amphibians and fish CCR3 clustered separately, and fish CCR3 formed a large cluster. PaCCR3 had the highest evolutionary correlation with that of rainbow smelt. Real-time quantitative PCR (qRT-PCR) analysis showed that the highest mRNA expression of PaCCR3 gene was observed in the MO/MФ from healthy ayu. The expression in liver, spleen and head kidney significantly up-regulated after V. anguillarum infection. Moreover, the mRNA expression of PaCCR3 was significantly up-regulated in ayu MO/MФ stimulated by V. anguillarum in vitro. The N-terminal extracellular peptide fragment of PaCCR3 was chemically synthesized, and its antiserum was obtained by immunizing New Zealand white rabbits. The Western blot analysis showed that the protein expression of PaCCR3 in ayu MO/MФ also increased significantly after V. anguillarum infection. Therefore, the mRNA and protein expression of PaCCR3 gene could be induced in response to pathogen infection. This study revealed for the first time that the mRNA and protein expression of PaCCR3 in response to V. anguillarum infection, and PaCCR3 may participate in the immune response by mediating the functional activity of MO/MФ, which can provide a new entry point for in-depth exploration of the function and mechanism of fish CCR3. |
| Key words: Plecoglossus altivelis CCR3 monocyte/macrophage (MO/MФ) Vibrio anguillarum expression analysis |
