鱼类免疫球蛋白D（IgD）是一类新近发现的免疫球蛋白类型，目前有关鱼类IgD免疫学功能相关研究仅在基因水平上略有涉及，而在蛋白水平几乎处于空白。本项目拟在前期克隆获得大菱鲆IgD编码全序列的基础上，分别利用牙鲆胚胎细胞系及大肠杆菌重组表达IgD重链δ恒定区，以此为免疫原研制抗大菱鲆天然IgD单克隆抗体。利用研制的单抗结合免疫学技术对大菱鲆天然IgD蛋白结构特征、分泌特性以及其在B淋巴细胞上及不同组织中的分布特点进行分析研究；在此基础上，利用流式细胞术及夹心ELISA技术分别构建IgD在细胞及蛋白水平上的免疫学检测方法，结合运用QPCR技术跟踪监测大菱鲆在利用注射及浸泡感染细菌性病原条件下IgD的免疫应答特点；同时，利用免疫诱导剂LPS及poly(I:C)刺激原代培养的淋巴细胞，利用免疫学及QPCR技术检测IgD的应答特点；从而阐释鱼类IgD在免疫监视、免疫防御应答及免疫信号传导中的功能特性

Immunoglobulin D (IgD) was recently found to be present in fish. Till now, IgD in fishes has mainly been studied at the genetic level, and very little is known about its fuctions in protein level. In this project， our previous cloned IgD cDNA of turbot was expressed in procaryotic and eukaryotic expression system, which then was employed as the immunogen to produc monoclonal antibodies (Mabs) against IgD. Using the Mabs against IgD, the protein structural characteristics of native IgD, secretoty featrues of IgD and the tissue and cellular distribution of IgD were investigated; Flow cytometry and sandwich ELISA technique were employed to develop the IgD detection method at cellular and protein levels respectively, which were combined with QPCR technique to investigate the IgD response against bacterial infection using injection or immersion routes; Meanwhile, following stimulated with LPS and poly(I:C), IgD variation in primary cultured leucocytes was investigated using immunological methods and QPCR technique. All the resultant data would reveal the mainly immunological functions of IgD in turbot.