Tang Xiaoqian, Qin Yinghui, Sheng Xiuzhen, Xing Jing, Zhan Wenbin
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, PR China.
Fish Shellfish Immunol. 2017 Apr;63:220-227. doi: 10.1016/j.fsi.2017.02.024. Epub 2017 Feb 20.
The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3ε of Japanese flounder (Paralichthys olivaceus) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3ε polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3ε of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3 T lymphocytes could be identified specifically by the mouse anti-rCD3ε polyclonal antibodies, which didn't cross-react with the sIgM lymphocytes. Immunohistochemistry showed that CD3 T lymphocytes could be detected in gill, skin, stomach, intestine, spleen, liver, head-kidney and mid-kidney. Flow cytometric analysis showed the percentages of CD3 T lymphocytes in the PBL, spleen lymphocytes (SL) and head-kidney lymphocytes (HKL) of Japanese flounder increased rapidly after immunization with formalin-inactivated Edwardsiella tarda, and reached their peak levels at 5th day with 12.6%, 9.7% and 8.7%, respectively, and then decreased gradually. These results suggested that CD3 T lymphocytes play important roles in mucosal and cell-mediated immunity, and the results would deepen our understanding on the roles of teleost T lymphocytes in the immune response.
CD3复合物是T淋巴细胞重要的细胞表面标志物，对于高等脊椎动物中T淋巴细胞的激活至关重要。在本研究中，牙鲆（Paralichthys olivaceus）的CD3ε在大肠杆菌BL21(DE3)中重组表达，并用作免疫原制备小鼠抗rCD3ε多克隆抗体，该抗体通过免疫共沉淀试验能够特异性识别牙鲆外周血淋巴细胞（PBL）膜蛋白中的一种20 kDa蛋白。质谱分析表明该20 kDa蛋白为牙鲆的天然CD3ε。流式细胞术分析和双重免疫荧光分析（DIFA）均显示，小鼠抗rCD3ε多克隆抗体能够特异性识别CD3 T淋巴细胞，且不与sIgM淋巴细胞发生交叉反应。免疫组织化学显示，在鳃、皮肤、胃、肠、脾脏、肝脏、头肾和中肾中均可检测到CD3 T淋巴细胞。流式细胞术分析显示，用福尔马林灭活迟缓爱德华氏菌免疫后，牙鲆PBL、脾脏淋巴细胞（SL）和头肾淋巴细胞（HKL）中CD3 T淋巴细胞的百分比迅速增加，并在第5天达到峰值水平，分别为12.6%、9.7%和8.7%，随后逐渐下降。这些结果表明，CD3 T淋巴细胞在黏膜免疫和细胞介导的免疫中发挥重要作用，该结果将加深我们对硬骨鱼T淋巴细胞在免疫应答中作用的理解。
