猪鼻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 hyorhinis to host cells

作　　者：纪燕[1,2] 熊祺琰[2] 韦艳娜[2] 马庆红[2] 白方方[2] 冯志新[2] 刘茂军[2] 邵国青[1,2] 

机构地区：[1]南京农业大学动物医学院,南京210095 [2]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,南京210014

出　　处：《中国人兽共患病学报》2014年第3期239-243,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31300155);江苏省自然科学基金(No.BK20130702)~~

摘　　要：目的猪鼻支原体是临床猪场的常见病菌,可引起猪多发性浆膜炎、肺炎、关节炎、中耳炎等慢性炎症,同时其与多种人类肿瘤有明显相关性,但其致病机理有待深入,其感染细胞机制的研究尚未明确,因此,本研究欲建立一种用间接免疫荧光技术检测猪鼻支原体黏附宿主细胞的方法。方法以猪鼻支原体和猪肾上皮细胞为研究对象,以兔抗猪鼻支原体纯化抗体为一抗,以FITC标记的羊抗兔IgG为二抗,通过反应条件的优化,建立猪鼻支原体黏附宿主细胞的间接免疫荧光检测方法(IFA)。结果最终确定最小黏附滴度为1∶107 CCU/mL,猪鼻支原体黏附PK15所需时间为6h以上,一抗的最佳工作浓度为1∶100,二抗的最佳工作浓度为1∶1 600。结论表明间接免疫荧光技术可以用来检测猪鼻支原体对宿主细胞的黏附作用。为猪鼻支原体的研究特别是感染细胞机制的体外研究提供基础方法,同时为疾病的诊断及疫苗的研发奠定基础。In the present study, an indirect immunofluorescence assay （IFA） was established for the detection of Mhr adherence to host cells. We used purified anti-Mhr IgG as the primary antibody and the fluorescein （FITC） isothioeyanated con- jugate goat anti-rabbit IgG as the secondary antibody. Optimization of reaction conditions was carried out, including the concen- tration of first antibody and second antibody, and then the minimum titer and the time of Mhr adhesion to cells. As a result, the optimum working concentration of anti-Mhr IgG and FITC-labeled goat anti-rabbit IgG was determined to be 1 ： 100 and 1 ： 1 600, respectively. The minimum titer and time of Mhr adhesion to the PK15 ceils was 1 × 10^7 CCU/mL and longer than 6 hours. The IFA established in this study provides the basic method, in particular, for the in vitro study of cellular mecha- nisms of infections caused by Mhr. In conclusion, our study outlines the basis for disease diagnosis and the development of Mhr vaccine.

关 键 词：猪鼻支原体 黏附 间接免疫荧光技术 

分 类 号：R375[医药卫生—病原生物学]