猪肺炎支原体P46蛋白单克隆抗体制备  

Preparation of Monoclonal Antibody against P46 Protein of Mycoplasma hyopneumoniae

作　　者：冯志新[1] 迟灵芝 刘茂军[1] 王海燕[1] 甘源[1] 吴叙苏[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]山东畜牧兽医职业学院,山东潍坊261061

出　　处：《江苏农业学报》2010年第2期320-324,共5页Jiangsu Journal of Agricultural Sciences

基　　金：公益性行业(农业)科研专项项目(200803020);中国博士后基金项目(20070421022);江苏省农业科技自主创新基金项目[CX(08)608]

摘　　要：P46蛋白是猪肺炎支原体的主要免疫优势蛋白,且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。制备针对该蛋白的特异性单抗能为猪肺炎支原体的检测及机理研究提供有效的资源。本研究取猪肺炎支原体全菌抗原免疫小鼠,用大肠杆菌表达的P46蛋白作为筛选抗原制备了2株特异性单克隆抗体1A4和3C11。结果显示,2株单抗ELISA效价最高可达1∶64 000。Western-blot结果表明,2株单抗均能与原核表达的P46蛋白及猪肺炎支原体全菌蛋白中46 000大小的蛋白发生特异性反应;2株杂交瘤细胞株经3个月冻存或连续传代3个月均能保持较高的滴度。本研究成功制备了2株针对猪肺炎支原体P46蛋白的特异、稳定且高滴度的单克隆抗体。P46 protein,the immunodominance protein of Mycoplasma hyopneumoniae（Mhp）,with no cross reaction with other mycoplasmas,is used as the target protein or gene for Mhp detection.The monoclonal antibody of P46 is more important for detection and pathogenic mechanism research of Mhp.In this study,the whole Mycoplasma hyopneumoniae protein was used as immunogen to immunize BALB/c mice for the preparation of monoclonal antibody.The P46 fusion protein expressed in E.coli was used to screen the monoclonal antibody against this specific antigen.Two strains of hybridoma cells,1A4 and 3G11 against P46 protein,were obtained through parallel screening with ELISA and Western blotting.And the strain 1A4 produced high ELISA titer of 1∶64 000.Two hybridoma cell lines could secrete highly efficient anti-P46 fusion protein antibody even after serial transfers or freezing for three months.

关 键 词：猪肺炎支原体 P46蛋白 单克隆抗体 

分 类 号：S852.62[农业科学—基础兽医学]