日本七鳃鳗MIF多克隆抗体的制备及应用

Preparation and application of Lampetra japonica MIF polyclonal antibody

目的以日本七鳃鳗(Lampetra japonica)类淋巴细胞为研究对象提取总RNA,通过RT-PCR方法获得七鳃鳗MIF基因对其进行生物信息学分析。方法将MIF基因构建到原核表达载体p ET-28a上,诱导表达并纯化MIF蛋白。以可溶表达的MIF蛋白免疫新西兰兔,应用间接ELISA方法和Western blot方法检测多克隆抗体。结果 ELISA检测抗体效价为1∶512 000;Western blot结果显示该多克隆抗体能特异性的结合重组和天然MIF蛋白;流式细胞术分析确定了19.36%的七鳃鳗类淋巴细胞表达MIF分子;激光共聚焦分析技术确定MIF分子主要定位于七鳃鳗类淋巴细胞的细胞浆中。结论重组MIF蛋白表达及多克隆抗体的成功制备,为七鳃鳗免疫功能方面的相关研究奠定了基础。

Macrophage migration inhibitory factor（MIF） mainly associated with phagocytic cell activation functions, including phagocytic cells adhesion, diffusion, phagocytosis and inhibition of apoptosis. In this experiment, we aimed to prepare Lampetra japonica MIF polyclonal antibodies. Total RNA was extracted from the Japanese lamprey lymphocyte, and then MIF gene was amplified by RT-PCR and analyzed by bioinformatics methods. In addition, MIF gene was successfully constructed to p ET-28 a prokaryotic expression vector, and then MIF protein was induced and purified. New Zealand rabbits were immunized using the purified MIF protein, and anti-MIF lamprey monospecific polyclonal antibody was purified by CNBr-activated Sepharose. ELISA showed that the titer of the prepared polyclonal antibody was 1 ∶512 000; Western blot indicated that the polyclonal antibody could specifically binding with recombinant or wild MIF protein; flow cytometry analysis identified 19.36% of lamprey lymphocyte expressing MIF molecules; while laser confocal analysis technique showed the MIF molecules were mainly in cytoplasm of lamprey lymphocytes. In conclusion, Japanese lamprey MIF protein and its polyclonal antibody have successfully prepared, which lay foundation for the lamprey MIF gene function research and other immune-related studies.