【作者】 孙峰；

【导师】 刘云；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2004， 硕士

【摘要】 牙鲆(Paralichthys olivaceus)是我国沿海网箱及池养的一个重要鱼种，目前病害日趋严重，如大量使用抗生素会危害人类的食品安全，而利用疫苗防治疾病具有明显的优越性。因此研究鱼类免疫系统的基本结构和功能，具有重要的理论意义和应用前景。制备针对免疫球蛋白的多克隆或单克隆抗体，作为研究工具，对免疫器官中产生抗体的组织和细胞进行定位，可以了解免疫器官在防御疾病过程中是如何发挥作用的，以便对应用疫苗奠定理论基础。制备多克隆或单克隆抗体的关键就是分离纯化出尽可能多的纯度较高的免疫球蛋白，为此本文对免疫球蛋白的纯化进行了探讨，并制备了多克隆抗体和应用免疫组化方法对牙鲆的主要免疫器官进行了研究。 运用两种方法对牙鲆的血清免疫球蛋白进行分离纯化，证明了Sephacryl S-200和HiTrap rProtein A Sepharose在纯化牙鲆血清免疫球蛋白的有效性，并纯化了一定量的牙鲆血清免疫球蛋白，同时测定了牙鲆血清免疫球蛋白的分子量为836kDa，其重链分子量为75.4kDa，具有分子量分别为29.9kDa和28.3kDa的两类轻链。 以提纯的免疫球蛋白为抗原免疫新西兰大白兔制备了兔抗牙鲆血清免疫球蛋白多克隆抗体，用免疫双扩散法检测多克隆抗体效价1：32，免疫斑点法检测多克隆抗体效价至少为1：2000以上。运用免疫印记法检测了兔抗牙鲆血清免疫球蛋白多克隆抗体的特异性，实验证明其能与牙鲆全血清中免疫球蛋白重链、轻链反应，而与人血清IgM无交叉反应。 利用自制的兔抗牙鲆血清免疫球蛋白多克隆抗体研究了产生免疫球蛋白的B淋巴细胞在牙鲆主要免疫器官中的分布。免疫组化方法检测出了牙鲆血液中产生免疫球蛋白的淋巴细胞，在牙鲆的头肾、体肾和脾的基质中都有产生免疫球蛋白的淋巴细胞存在，从数量上看头肾中最多，脾中次之，体肾中最少。 研究牙鲆黏膜样淋巴组织结果显示在牙鲆的后肠中存在大量产生免疫球蛋白的淋巴细胞，主要分布于后肠的固有层和粘膜下层中，在粘膜层和粘膜层表面也偶有极少量的产生免疫球蛋白的淋巴细胞出现。更多还原

【Abstract】 Flounder, Paralichthys olivaceus, a species of high commercial value and reared in costal China, has been attacked by many diseases nowadays. Vaccine may play an important role to enhance the healthy aquiculture while abuse antibiotics increases hazards to human health. In order to produce and popularize use of vaccine for fish, study fish immune system becomes compulsory. To attain more comprehensive understanding of flounder immune system, it is necessary to characterise and localise lymphoid cells within the different lymphoid organs. Use polyclonal antibody or monoclonal antibody as a tool will benefit this kind of study.This paper purified serum immunoglobulin of flounder by using Sephacryl-200 gel column chromatography after ammonium sulfate precipitation and HiTrap rProtein A Sepharose affinity chromatography. The results show that former method by which enough immunoglobulin for further study attained maybe more effective than latter. The molecular weight of flounder serum immunoglobulin is 836 kDa and that of heavy chain is 75.4 kDa, while that of light chains are 29.9 kDa and 28.2 kDa respectively.Meanwhile, polyclonal rabbit antibody against flounder serum immunoglobulin was produced. The potency determined by double immunodiflusion was 1: 32 and a few more than 1:2000 by immunodotting. The Western-blotting illuminated the antibody had immunological activity by reaction with heavy chain and light chains of flounder serum immunoglobulin and no cross reaction with that of human.The polyclonal rabbit antibody against flounder serum immunoglobulin was used as a marker for immunohistochemical detection of B cells. The present investigation reports there are two kinds of lymphoid cells which are Ig-bearing and non Ig-bearing cells existing in flounder blood. This antibody reacted with Ig-bearing lymphocytes in paraffin-embeddedsections from kidney and spleen which are main lymphoid organs in flounder. The rank of Ig-bearing lymphocytes number is head kidney, spleen and trunk kidney.In addition, hindgut has also been analysed, as studies have suggested that numerous lymphoid cells occur at this location in the lamina propria and the submucosa. The Ig-bearing cells are also found in the epithelial layer occasionally.更多还原