草鱼抗菌肽Hepcidin过表达对拟态弧菌生长和NF-κB通路的影响

Overexpression of grass carp (Ctenopharyngodon idella) antimicrobial peptide Hepcidin activates NF-κB pathway and its effect on the growth Vibrio mimicus

摘要:

【背景】拟态弧菌(Vibrio mimicus)是一种严重危害水产养殖业的病原菌，可引起鱼类弧菌病，目前使用抗生素防治该病存在较多弊端。【目的】在细胞水平探讨过表达草鱼抗菌肽Hepcidin (CiHep)对拟态弧菌生长和NF-κB通路的影响。【方法】构建重组真核表达质粒pEGFP-N1-Cihep和pcDNA3.1(+)-Cihep。将pEGFP-N1-Cihep转染草鱼肾细胞系(Ctenopharyngodon idellakidney，CIK)，通过免疫荧光法和Western blotting检测转染后不同时间融合蛋白EGFP-CiHep的表达情况。进一步检测转染pcDNA3.1(+)-Cihep后的CIK细胞培养上清液对拟态弧菌生长的影响，以及转染细胞内NF-κB通路激活关键基因和下游免疫相关基因的转录水平。【结果】融合蛋白EGFP-CiHep在CIK细胞内能够良好表达，且以转染后48 h的表达量最高；转染后的细胞培养上清液能够显著抑制拟态弧菌生长；过表达CiHep可显著改变NF-κB通路激活关键基因和下游免疫相关基因的转录水平。【结论】 CiHep具有抑制拟态弧菌生长和激活NF-κB通路的作用。研究结果为鱼源Hepcidin的开发应用提供了理论依据，有望实现从激活NF-κB通路正向调控抗菌肽表达的新角度防治鱼类拟态弧菌病。

Abstract:

[Background] Vibrio mimicus is a pathogenic bacterium that severely impacts aquaculture. The application of antibiotics to the control of vibriosis caused by this pathogen leads to drug resistance, environment pollution, and food safety problems. [Objective] To explore whether overexpression of the antimicrobial peptide Hepcidin (CiHep) from Ctenopharyngodon idella affects V. mimicus growth and NF-κB pathway at the cellular level. [Methods] The recombinant plasmids pEGFP-N1- Cihep and pcDNA3.1(+)- Cihep were constructed for eukaryotic expression. The expression of the fusion protein EGFP-CiHep was detected by immunofluorescence assay and Western blotting at different time points post transfection of pEGFP-N1- Cihep in C. idella kidney (CIK) cells. Then, the effect of the supernatant of the cells transfected with pcDNA3.1(+)- Cihep on the growth of V. mimicus was examined. The transcription levels of the key genes for NF-κB pathway activation and the downstream immunity-related genes in the transfected cells were measured. [Results] EGFP-CiHep was well expressed in the CIK cells and reached the highest expression level 48 h post transfection. The supernatant of the transfected cells significantly inhibited the growth of V. mimicus. CiHep overexpression significantly altered the transcription levels of key genes for NF-κB pathway activation and downstream immunity-related genes. [Conclusion] CiHep can inhibit V. mimicus growth and activate NF-κB pathway. Our results provide a theoretical basis for developing fish-derived Hepcidin and a new perspective on regulation of antimicrobial peptide expression by activating NF-κB pathway for prevention and treatment of fish vibriosis.