牙鲆(Paralichthys olivaceus)红细胞单克隆抗体与五种养殖鱼类血细胞的交叉反应

CROSS REACTION OF MONOCLONAL ANTIBODIES AGAINST FLOUNDER PARALICHTHYS OLIVACEUS WITH BLOOD CELLS OF OTHER FIVE FISH SPECIES

以牙鲆血细胞为抗原免疫Balb/c小鼠 ,选用聚乙二醇 (PEG)作为细胞融合剂 ,将免疫的小鼠脾细胞与P3X63Ag8U1骨髓瘤细胞融合 ,筛选、克隆出 3株生产抗牙鲆红细胞单克隆抗体 (Mabs)的杂交瘤细胞 ( 1C7、2B6、3A1 )。应用免疫荧光抗体法 (IFAT)研究 3株单抗与 5种常见养殖鱼类 (大菱鲆、花鲈、真鲷、许氏平、鲫鱼 )红细胞的交叉反应 ,结果显示 ,3株单抗与5种鱼类红细胞有不同程度的阳性反应 ;应用Westernblotting法分析单抗识别的蛋白分子量 ,结果显示 ,单抗 1C7与大菱鲆血细胞结合的蛋白分子量为 5 2kD、47kD、45kD、32kD、2 9kD、2 7kD ,与花鲈、许氏平结合的蛋白分子量是 5 2kD、2 9kD ,与真鲷结合的为 2 9kD ,与鲫鱼结合的为44kD、2 9kD、2 8kD ;单抗 2B6与大菱鲆、花鲈血细胞结合的蛋白带分子量是 30kD、2 8kD ,与真鲷结合的为 30kD ,与许氏平结合的为 2 9kD、2 8kD ;单抗 3A1没有Westernblotting反应。结果表明 ,这 6种鱼红细胞存在相同或相似的抗原决定簇 ,蛋白成分具有相似性。

Monoclonal antibodies (Mabs) have proven to be powerful tools in the characterization of mammalian blood cells in terms of their ontogeny, interrelationships, and functions. Therefore, it is natural that workers with non-mammalian such as fish blood cells have attempted to use similar approaches in the development of much needed methods for the characterization of these cells. In order to study the molecular mechanism of immunology on fish blood cells, monoclonal antibodies were raised. Four-week old Balb/c mice were immunized 4 times within 4 weeks with blood cell of healthy flounder, Paralichthys olivaceus. Three days after the last immunization, spleens of the immunized mice were dissected into cells and then fused with P3-X63-Ag8U1 myeloma cell line using 50% polyethylene glycol (PEG) as fusogen. The fused cells were cultured in HAT-GIT selecting medium for about 2 weeks. The survival cells (hybridoma cells) were cultured in GIT. Mediums of hybridoma cells were detected by indirect immunofluorescence assay test (IFAT). Many positive hybridomas were found and 3 of them were cloned because of secreting high titer antibodies. As they were cloned 3 times continuously, it could be verified that the antibodies raised by these hybridoma cell lines were monoclonal. Then the monoclonal antibodies were used to cross-react with the blood cells from other 5 fish species (Scophthalmus maximus, Lateolabrax japonicus, Pagrosomus major, Sebastes schlegeli, Carassius auratus) by indirect immunofluorescence assay test and using Western blotting. Results of this treatment yielded 3 stains of hybridoma cell lines (1C7, 2B6, 3A1) able to secrete monoclonal antibodies against flounder erythrocyte. The cross reaction showed that these monoclonal antibodies reacted with other 5 fish erythrocytes in different degrees: Mab 1C7 reacted with all of the 5 fish erythrocytes. Mab 2B6 reacted with erythrocytes of S. maximus, L. japonicus, P. major and S. schlegeli. Mab 3A1 reacted with erythrocytes of S. maximus and L. japonicus. Further experiments by Western blotting showed Mab 1C7 was able to recognize the protein of S. maximus blood cell whose molecule weight were 52, 47, 45, 3, 29 and 27kD; the protein of L. japonicus and S. schlegeli blood cell of 52 and 29kD; the protein of P. major blood cell of 29kD; the protein of C. auratus blood cell of 44, 29 and 28kD. Mab 2B6 recognized the protein of S. maximus and L. japonicus blood cell whose molecule weight were 30 and 28kD; the protein of P. major blood cell of 30kD; the protein of S. schlegeli blood cell of 29 and 28kD. Mab 3A1 could not recognize the protein by Western blotting. These experiments indicate the presence of the same epitopes on the blood cells of these species of fish. The monoclonal antibodies are ready for use in ontogeny research of fish erythrocyte.