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。

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 Cihepcidin 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. Subsequently, fluorescence co-localization, co-immunoprecipitation and Western blot were used to identify the intracellular 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 spatio-temporal expression patterns in each examined tissue at different time points post-infection, while the expression 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-regulated 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.

S 942.1

国家自然科学基金 (31672698)；安徽农业大学研究生创新基金 (2020ysj-29)