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目的进一步认识尼罗罗非鱼免疫球蛋白的结构与功能。方法采用RACE-PCR技术克隆免疫球蛋白M重链c DNA全长,并将其CH2-4与PSUMO构建重组表达载体,诱导表达、纯化后,免疫小鼠获得抗血清,用ELISA及免疫印迹检测抗体的特性。结果经分析该序列含1919nt,编码514个氨基酸。纯化蛋白经SDS-PAGE电泳显示得到了相对分子质量为36 200的目的蛋白。ELISA及免疫印迹检测表明抗血清能特异识别尼罗罗非鱼血清中IgM。结论成功获得的尼罗罗非鱼IgM cDNA序列及其抗体制备,为其免疫系统的研究及相关免疫检测奠定了基础。
Objective To further understand the structure and function of Nile tilapia immunoglobulin. Methods The full-length cDNA of immunoglobulin M heavy chain was cloned by RACE-PCR, and its recombinant plasmid was constructed by the expression of CH2-4 and PSUMO. The recombinant plasmid was induced and purified. The antiserum was obtained from the mice by ELISA and Western blotting Antibody characteristics. The results of analysis of the sequence contains 1919nt, encoding 514 amino acids. Purified protein SDS-PAGE electrophoresis showed that the relative molecular mass of 36 200 of the target protein. ELISA and Western blot showed that antiserum could specifically recognize IgM in serum of Nile tilapia. Conclusion The IgM cDNA sequence of Nile tilapia and its antibody preparation successfully laid the foundation for its immune system research and related immunoassay.