首页|草鱼TAB2与TAK1蛋白互作鉴定及其对两种抗菌肽基因表达的影响

草鱼TAB2与TAK1蛋白互作鉴定及其对两种抗菌肽基因表达的影响

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为了探究草鱼TAB2(CiTAB2)与CiTAK1 能否互作及其对 2 种草鱼抗菌肽基因(Cihepcidin与Ciβ-defensin1)表达的影响，实验首先采用实时荧光定量PCR(qPCR)方法分析拟态弧菌感染后Citab2 和Citak1 在草鱼免疫相关组织中的时空表达模式。然后利用荧光共定位、免疫共沉淀及Western blot技术鉴定CiTAB2 与CiTAK1 在细胞内共定位及相互作用情况。最后将过表达质粒pEGFP-N1-Citak1 与pEGFP-N1-Citab2 共同转染草鱼肾细胞(CIK细胞)，检测Cihepcidin与Ciβ-defensin1 的相对mRNA表达水平。结果显示，拟态弧菌感染能够显著改变Citab2 和Citak1 的相对表达水平，前者于感染后不同时间在各检测组织中表现出不同的时空表达模式，而后者均呈现先上调后下调的表达模式;荧光显微镜下观察到 CiTAB2 与 CiTAK1 共定位于转染后的 HEK293T和 CIK细胞的胞质中，且在HEK293T细胞内能够形成 CiTAB2-CiTAK1 蛋白复合物;共同过表达 CiTAB2 与CiTAK1 后，CIK细胞内Cihepcidin与Ciβ-defensin1 的相对mRNA表达水平在各检测时间点均显著上调。结果表明，CiTAB2 与CiTAK1 存在互作关系且二者互作能够促进上述两种抗菌肽的转录表达。本研究从蛋白互作调控抗菌肽表达的角度为防治鱼类弧菌病提供了新策略。
Identification of the interaction between TAB2 and TAK1 proteins from Ctenopharyngodon idella and effect of their interaction on the expression of two antimicrobial peptides genes
To investigate whether grass carp(Ctenopharyngodon idella)TAB2(CiTAB2)can interact with CiTAK1 and the effect of their interaction on the expression of two antimicrobial peptides(AMPs)genes Cihep-cidin and Ciβ-defensin1,the temporal and spatial expression patterns of Citab2 and Citak1 in the immune-related tissues of grass carp after Vibrio mimicus infection were firstly analyzed by qPCR in the present study.Sub-sequently,fluorescence co-localization,co-immunoprecipitation and Western blot were used to identify the intra-cellular co-localization and interaction between CiTAB2 and CiTAK1 proteins.Finally,the mRNA expression levels of Cihepcidin and Ciβ-defensin1 were examined after the overexpression plasmids pEGFP-N1-Citak1 and pEGFP-N1-Citab2 were co-transfected into CIK cells.The results showed that V.mimicus infection significantly altered the relative mRNA expression levels of Citab2 and Citak1.Among them,the former showed different spa-tio-temporal expression patterns in each examined tissue at different time points post-infection,while the expres-sion pattern of the latter was up-regulated then down-regulated in all examined tissues.CiTAB2 and CiTAK1 were co-localized in the cytoplasm of both HEK293T and CIK cells observed under a fluorescence microscope,and the CiTAB2-CiTAK1 protein complex could be formed in HEK293T cells post-transfection.After co-overexpression of CiTAB2 and CiTAK1,the mRNA expression levels of Cihepcidin and Ciβ-defensin1 were significantly up-reg-ulated in the CIK cells at each test time point.These results indicated that CiTAB2 could interact with CiTAK1 and their interaction was able to promote the transcriptional expression of these two AMPs,which provides a new strategy for the control of fish vibriosis from the perspective of protein interaction.

Ctenopharyngodon idellatransforming growth factor-β-activated kinase-1(TAK1)TAK1 binding proteins 2(TAB2)protein interactiongene expression of AMPs

杨文飞、郭佳静、赵文平、李槿年

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安徽农业大学动物科技学院,安徽合肥 230036

草鱼 转化生长因子-β激活激酶1(TAK1) TAK1结合蛋白2(TAB2) 蛋白互作 抗菌肽基因表达

国家自然科学基金安徽农业大学研究生创新基金

316726982020ysj-29

2024

10.11964/jfc.20210913077

水产学报
中国水产学会

水产学报

CSTPCD北大核心
影响因子：1.148
ISSN：1000-0615
年,卷(期)：2024.48(2)
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