溶藻弧菌诱导红笛鲷分泌型/膜型免疫球蛋白基因的表达及定位

We have successfully completed the research content of the project under the fund of NSFC. Results showed the non-specific immune reactions were absolutely dominant in the larval of red snapper, in which the related genes encoding heat shock proteins (HSP10, HSP90, HSC70) and non-specific cytotoxic cell receptor protein 1 (NCCRP-1) were significantly up-regulated after vaccinated by inactivated V. alginolyticus. On the other hand, the recombination activating genes (rag) were also up-regulated under the vaccination of V. alginolyticus, which indicates that the specific immune response in fish larval can be induced . In the project implementation period, 5 paper recorded by SCI and 2 paper recorded by CSCD were published with the acknowledgement for the first fund of the project. When referring to personnel training, there are one PhD and four Master graduated with the dissertation related to the project. Moreover, Two people in the project group were funded by the China Scholarship Council and went to Norway and the United States as visiting scholars. In the present applied project, the molecular basis of the specific immune response of fish——the immunoglobulin will be focused and studied The expression of genes encoding the secretory and membrane-type IgM, IgD and IgZ in various tissues and organs of red snapper will be determined after induced by Vibrio alginolyticus, and the subcellular localization of the membrane-type Ig by GFP-labeled plasmid as well as the direct analysis of Ig positive cells of various types (or type) Ig will be clarified using in situ hybridization method. The present work will be useful in interpretation the function of various types (or type) Ig in the antibacterial immunity of fish.

在青年基金资助下，顺利完成了项目内容，证明了非特异性免疫在红笛鲷仔鱼抵御细菌感染中处于绝对主导地位，其中HSP家族基因（HSP10、HSP90、HSC70）和非特异性细胞毒性细胞受体蛋白1（NCCRP-1）基因均会在弧菌诱导下大量表达，而重组激活基因（rag）在弧菌诱导下的上调表达则表明特异性免疫应答在仔鱼期是可以诱导的。本项目执行期间计发表SCI论文5篇，中文核心期刊2篇，培养博士生1人，硕士生4人，项目组中有2人获国家留学基金委资助作访问学者。本次申请项目是在上一个基金项目的基础上，拟从特异性免疫应答的分子基础——免疫球蛋白入手，研究灭活溶藻弧菌诱导后，红笛鲷分泌型和膜型IgM、IgD、IgZ基因在各组织器官表达的先后和强度，并利用GFP标记质粒对膜型Ig进行亚细胞定位，利用原位杂交技术对各类（或型）Ig阳性细胞进行分析，进而从分子角度对各类（或型）Ig在抗菌免疫中的作用进行阐释。

本项目拟从特异性免疫应答的分子基础——免疫球蛋白（Ig）入手，研究细菌诱导后，鱼类分泌型和膜型免疫球蛋白基因在头肾、脾脏、胸腺、肠道、肝脏、肾脏、心脏、脑、性腺、肌肉、皮肤、鳃等组织器官中表达的先后和强度，并利用原位杂交技术对各类（或型）Ig 阳性细胞进行组织定位，进而从分子角度对各类（或型）Ig 在抗菌免疫中的作用进行阐释。通过为期一年的初步研究，项目组成功获得了红笛鲷sIgZ和mIgZ、罗非鱼mIgD和sIgM的全基因序列，阐明了上述四种免疫球蛋白与其它硬骨鱼类免疫球蛋白的同源性；对上述四种免疫球蛋白在鱼类抗菌免疫过程中的表达时间序列进行了分析，首次发现mIgD的表达可能受IgM的影响和调控的现象；对上述四种免疫球蛋白在鱼类组织间的表达差异进行了分析，阐明了红笛鲷sIgZ和mIgZ、罗非鱼mIgD和sIgM首次达到高峰的组织均属于粘膜免疫系统，提示它们可能均参与粘膜免疫应答。项目研究结果有利于揭示鱼类免疫球蛋白在抗菌免疫应答中的变化规律，对计划书所提出的科学问题给出了满意的答案。截止到结题日，项目发表科技论文6篇，其中SCI收录1篇，中文期刊5篇；另外，已修回SCI论文1篇，整理待发表英文论文1篇。培养硕士生3名，博士生1名，达到了项目计划书所提出的目标。