摘要
P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.
P65蛋白是猪肺炎支原体的主要免疫优势蛋白,与其他支原体无交叉反应,常作为猪肺炎支原体检测的靶蛋白。本实验应用原核表达的重组P65蛋白免疫小鼠,用Mhp全菌蛋白和P65蛋白筛选,制备了1株特异性单克隆抗体3G12。鉴定结果表明,该株单克隆抗体可与P65蛋白和Mhp全菌蛋白反应,采用间接ELISA法测得3G12细胞培养上清与P65蛋白反应抗体的效价为1∶12 800,与全菌蛋白反应抗体的效价为1∶3 200;3G12腹水与P65蛋白反应的抗体效价为1∶4 000 000以上,与168全菌蛋白反应的抗体效价为1∶20 000以上,细胞经长期体外培养和冻存后复苏能稳定分泌抗体。本研究成功获得到1株针对P65和Mhp全菌的单克隆抗体,为猪肺炎支原体致病机制和检测方法的进一步研究提供了基础。
基金
Supported by National Natural Science Foundation of China(31100136,3111339)
Independent Innovation Fund of Agricultural Science and Technology of Jiangsu Province[CX(13)3066]~~
