尼罗罗非鱼 CD2AP 的分子克隆、鉴定及其在哈维氏弧菌感染应答中的表达特征，并与 CD2 胞质段相互作用。
Molecular cloning, characterization and expression profiles of CD2AP in Nile tilapia (Oreochromis niloticus) responding to Streptococcus agalactiae infection and interaction with CD2 cytoplasmic segment.
机构信息
Shenzhen Institute of Guangdong Ocean University, Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, 518120, Guangdong, China; Fisheries College of Guangdong Ocean University, Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Zhanjiang, China.
Shenzhen Institute of Guangdong Ocean University, Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, 518120, Guangdong, China; Fisheries College of Guangdong Ocean University, Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Zhanjiang, China; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
出版信息
Fish Shellfish Immunol. 2020 Jun;101:205-215. doi: 10.1016/j.fsi.2020.03.054. Epub 2020 Apr 1.
The interaction between CD2-associated protein (CD2AP) and CD2 plays a vital role in lymphocyte adhesion and T cells activation in mammals. In this study, a CD2AP gene (GenBank accession number: MK579862; designated as On-CD2AP) was identified from tilapia (Oreochromis niloticus). Sequence analysis showed that On-CD2AP protein shares high similarity with mammals, including three Src homology 3 (SH3) domains, a section of poly proline motif and a coiled coil region. Transcription levels of On-CD2AP were detected in nine tissues of healthy Nile tilapia, and the highest expression levels were detected in the spleen and gill. On-CD2AP were significantly up-regulated in thymus, head kidney and brain after infected by Streptococcus agalactiae, as well as in head kidney leukocytes (HKLs) with LPS and LTA stimulation. Moreover, a section conserved pro-rich motif that are responsible for binding of CD2 to CD2AP were found in the CD2 cytoplasmic sequence of Nile tilapia (On-CD2C). A weak interaction between On-CD2AP and On-CD2C was proved by yeast two-hybrid assay. In addition, the recombinant proteins of CD2AP-His (rOn-CD2AP-His) and GST-CD2C (GST-rOn-CD2C) were obtained through prokaryotic expression system. His pull-down assay showed that rOn-CD2AP-His and GST-rOn-CD2C could bind to each other. These findings indicate that CD2AP is crucial in immune response during S.agalactiae infection, and the mechanism of interaction between CD2AP and CD2 is conservative in Nile tilapia.
CD2 相关蛋白（CD2AP）与 CD2 之间的相互作用在哺乳动物淋巴细胞黏附和 T 细胞激活中起着至关重要的作用。本研究从罗非鱼（Oreochromis niloticus）中鉴定出 CD2AP 基因（GenBank 登录号：MK579862；命名为 On-CD2AP）。序列分析表明，On-CD2AP 蛋白与哺乳动物具有高度相似性，包括三个Src 同源 3（SH3）结构域、一段多脯氨酸基序和一个卷曲螺旋区。检测了健康尼罗罗非鱼 9 种组织中 On-CD2AP 的转录水平，发现其在脾脏和鳃中的表达水平最高。在感染无乳链球菌后，尼罗罗非鱼胸腺、头肾和脑中的 On-CD2AP 表达显著上调，LPS 和 LTA 刺激后头肾白细胞（HKLs）中的 On-CD2AP 表达也上调。此外，在尼罗罗非鱼 CD2 的胞质序列中发现了一段保守的富含脯氨酸的基序，该基序负责 CD2 与 CD2AP 的结合（On-CD2C）。酵母双杂交实验证明了 On-CD2AP 与 On-CD2C 之间存在较弱的相互作用。此外，通过原核表达系统获得了 CD2AP-His（rOn-CD2AP-His）和 GST-CD2C（GST-rOn-CD2C）的重组蛋白。His 下拉实验表明 rOn-CD2AP-His 和 GST-rOn-CD2C 可以相互结合。这些发现表明，CD2AP 在无乳链球菌感染期间的免疫反应中至关重要，并且 CD2AP 与 CD2 之间的相互作用机制在尼罗罗非鱼中是保守的。
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