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

作　　者：赵文平 岳小真 常娇娇 李槿年[1] ZHAO Wenping;YUE Xiaozhen;CHANG Jiaojiao;LI Jinnian(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,Anhui,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《微生物学通报》2022年第11期4786-4797,共12页Microbiology China

基　　金：国家自然科学基金(31672698)。

摘　　要：【背景】拟态弧菌(Vibriomimicus)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(Ctenopharyngodonidellakidney,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。[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.

关 键 词：草鱼抗菌肽Hepcidin 拟态弧菌 抗菌作用 NF-ΚB通路 

分 类 号：S943[农业科学—水产养殖]