猪肺炎支原体DnaK蛋白质单抗的制备与鉴定  

Production and identification of monoclonal antibodies against DnaK protein of Mycoplasma hyopneumoniae

作　　者：缪芬芳[1,2] 王海燕[1] 白昀[1] 刘茂军[1,2] 冯志新[1] 熊祺琰[1] 白方方[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2012年第6期1361-1366,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31001080);江苏省农业自主创新基金项目[CX(11)2059]

摘　　要：为了获得针对猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)DnaK蛋白质的单克隆抗体,利用Mhp全菌抗原免疫小鼠,采用杂交瘤技术制备Mhp DnaK蛋白质的单抗。获得了3株可稳定分泌特异性抗体的杂交瘤细胞株,分别命名为3A8、3E2及5D10。3株单抗的Ig重链均为IgG1,轻链均为κ链。Western Blot分析结果显示,3A8与猪鼻支原体和Mhp均有反应,3E2和5D10只与Mhp反应,同时3E2和5D10能与Mhp 168株105代、J株、临床分离株XLW-1、XLW-2、XLW-3、XLW-4、AH、WX产生特异性反应。采用间接ELISA测得3株单抗腹水与DnaK重组蛋白质及Mhp 168全菌蛋白质反应效价均为1∶1 000 000。单抗3A8为针对Mhp和猪鼻支原体(Mycoplasmahyorhinitis,Mhr)共同抗原表位的单抗;3E2和5D10为猪肺炎支原体特异性抗体,且抗体效价高。To obtain the monoclonal antibodies of Mycoplasma hyopneumoniae （Mhp） , Mhp 168 strain was used as immunogen to immunize BALB/c mice. With the hybridoma technology, three hybridoma cell lines which steadily secret monoclonal antibody against DnaK protein were obtained, separately named 3A8, 5DlO and 3E2. The isotype of the 3E2, 3A8 and 5DlO was IgG1, K chain. Western blot showed that all of the McAbs could react with Mhp168 isolates and the DnaK recombinant protein, and had no reaction activity with vector protein and Mycoplasma hyorhinitis （Mhr） except for 3A8. The McAbs of 3E2 and 5D10 also specifically reacted with 168 strain F105, J strain, XLW-1 isolate, XLW-2 isolate, XLW-3 isolate, XLW-4 isolate, AI-I isolate, and WX isolate of Mhp. Indirect ELISA revealed the titers of the ascites of thethree monoclonal antibodies against DnaK recombinant protein and Mhp 168 protein were 1 ： 1 000 000. 3A8 was the monoclonal antibody which reacted with the common antigen epitopes of Mhp and Mhr; 3E2 and 5D10 were the specific monoclonal antibodies against Mhp, both of them with high titers.

关 键 词：猪肺炎支原体 DnaK蛋白质 单克隆抗体 

分 类 号：S852.62[农业科学—基础兽医学]