猪肺炎支原体黏附宿主细胞间接免疫荧光检测方法的建立  

Establishment of an indirect immunofluorescence assay for detection of adherence of Mycoplasma hyopneumoniae to host cells

作　　者：车巧林[1] 熊祺琰[1] 冯志新[1] 张旭[1] 刘茂军[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

出　　处：《江苏农业学报》2012年第5期1069-1073,共5页Jiangsu Journal of Agricultural Sciences

基　　金：中国博士后面上项目(2012M510175);国家自然科学基金项目(31100136)

摘　　要：为了建立一种用间接免疫荧光技术检测猪肺炎支原体黏附宿主细胞的方法。以抗猪肺炎支原体单克隆抗体为一抗,异硫氰酸荧光素(FITC)标记的羊抗小鼠IgG为二抗,通过对一抗、二抗工作浓度的优化确定合适的抗体浓度;通过以1×107CCU/ml猪肺炎支原体与PK15细胞作用不同时间,确定其黏附细胞需要的时间;通过不同滴度的猪肺炎支原体与PK15细胞37℃作用4 h,确定其黏附细胞需要的滴度。猪肺炎支原体单克隆抗体P463G11的最适工作浓度为1∶1 000倍稀释,FITC-羊抗小鼠IgG的最适工作浓度为1∶200倍稀释,猪肺炎支原体黏附PK15细胞需要的时间为4 h以上,黏附PK15细胞需要的滴度为1×105CCU/ml以上。表明间接免疫荧光技术可以用来检测猪肺炎支原体对宿主细胞的黏附作用。This study was to establish an indirect immunofluorescence assay for detecting Mycoplasma hyopneumoniae adherence to host cells.Anti-Mycoplasma hyopneumoniae monoclonal antibody（McAb） was used as the primary antibody,and fluorescein isothiocyanate（FITC） labeled goat anti-mouse IgG was used as the second antibody.The optimal working concentrations of antibodies were determined via optimizing consistence of the primary antibody and second antibody.The adhesion time was determined by the time needed for M.hyopneumoniae of 1×107 CCU/ml to adhere PK15 cells.The adhesion titer was determined through the adherence of M.hyopneumoniae with different titers to PK15 cells at 37 ℃ for 4 h.The optimum working concentrations of anti-M.hyopneumoniae McAb P463G11 and FITC-labeled goat anti-mouse IgG were 1∶1 000 and 1∶200,respectively.The time and titer of M.hyopneumoniae adhesion to the PK15 cells were above 4 h and 1×105 CCU/ml.The results indicated indirect immunofluorescence assay could be used to detect M.hyopneumoniae adhesion to host cells.

关 键 词：猪肺炎支原体 黏附 间接免疫荧光 

分 类 号：S828.89[农业科学—畜牧学]