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作 者:田丁 蒋飞 陈春秀 李珂珂 卞柄杰 吴志新[1,2] 陈孝煊 TIAN Ding;JIANG Fei;CHEN Chun-Xiu;LI Ke-Ke;BIAN Bing-Jie;WU Zhi-Xin;CHEN Xiao-Xuan(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Hubei Engineering Research Center for Aquatic Animal Diseases Control and Prevention,Wuhan 430070,China)
机构地区:[1]华中农业大学水产学院,武汉430070 [2]湖北省水生动物病害防控工程技术研究中心,武汉430070
出 处:《水生生物学报》2023年第4期609-616,共8页Acta Hydrobiologica Sinica
基 金:国家自然科学基金(31972819)资助。
摘 要:为探讨树突状细胞(Dendritic cells,DCs)表面重要的表型分子CD8α和CD207在草鱼(Ctenopharyngodon idella)DCs抗细菌免疫应答中的作用,实验从草鱼DCs的cDNA中扩增CD8α和CD207胞外区,构建重组表达质粒pET-32a-CD8α/CD207,并转化到感受态细胞Transetta(DE3),表达纯化后制备CD8α和CD207多克隆抗体。利用qPCR、Western Blot及流式细胞术揭示草鱼CD8α和CD207在抗细菌免疫过程中发挥的功能。结果显示,制备的草鱼CD8α和CD207多克隆抗体既可以识别原核表达的重组蛋白,也能识别鱼体内和培养细胞的内源性蛋白。从功能上看,灭活嗜水气单胞菌处理草鱼DCs后,在24h内,CD8α和CD207的表达量均有显著上调(P<0.05);且CD8α和CD207分子在草鱼DCs呈递抗原刺激混合淋巴细胞增殖这一过程中发挥重要作用。因此,草鱼DCs表现出与哺乳动物相似的保守免疫表型和功能,研究结果为阐明草鱼DCs介导的适应性免疫调节机制奠定基础。CD8αand CD207 are important phenotypic molecules on Dendritic cells(DCs),which play important roles in DCs-mediated adaptive immunity.To investigate their role in the anti-bacterial immune response of grass carp(Ctenopharyngodon idella)DCs,the extracellular regions of CD8αand CD207 were amplified from the cDNA of grass carp DCs,and the recombinant expression plasmid pET-32a-CD8α/CD207 was constructed and converted to competent cell Transetta(DE3),where it was expressed and purified to produce polyclonal antibodies against CD8αand CD207.The functions of grass carp CD8αand CD207 in the anti-bacterial immune process were revealed by qPCR,Western Blot and flow cytometry.The results showed that the prepared polyclonal antibodies recognized both prokaryotic expressed recombinant proteins and endogenous proteins at the individual and cellular levels.Functionally,after treatment with inactivated Aeromonas hydrophila to grass carp DCs,the expression levels of CD8αand CD207 were significantly upregulated within 24h(P<0.05).Besides,the molecules of CD8αand CD207 play an important role in the process that the antigen presented by grass carp DCs stimulates the proliferation of mixed lymphocytes.Therefore,grass carp DCs exhibits a conservative immunophenotype and function similar to those in mammals,and the results of the study will provide a basis for elucidating the adaptive immune regulation mechanism mediated by grass carp DCs.
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