山羊支原体山羊肺炎亚种黏附山羊气管上皮细胞间接免疫荧光检测方法的建立

Establishment of an indirect immunofluorescence assay for detection of Mycoplasma capricolum subsp.capripneumoniae adhesion to goat tracheal epithelial cells

以山羊支原体山羊肺炎亚种(Mccp)M1801株和山羊气管上皮细胞(GTC)为研究对象,通过优化Mccp接种滴度、孵育时间及一抗、二抗工作浓度,建立Mccp黏附宿主细胞的间接免疫荧光检测方法。确定Mccp的最适黏附滴度为1.5×10^(7)~7.0×10^(7)copies/μL,黏附时间为2 h,一抗最适稀释度为1∶100,二抗最适稀释度为1∶100。用本试验建立的间接免疫荧光方法证实Mccp人工感染山羊血清可阻断其黏附GTC的作用,为Mccp感染的检测、黏附因子的鉴定及黏附机制的研究提供了有效技术手段。

Mycoplasma capricolum subsp.capripneumoniae(Mccp)M1801 strain and goat tracheal epithelial(GTC)cells were used.The indirect immunofluorescence assay(IFA)for Mccp adhesion to host cells was established by optimization of reaction conditions,including the inoculation titer and time of Mccp adhesion to cells,as well as working concentration of primary and secondary antibodies.Finally,it was determined that the optimal adhesion concentration of Mccp to GTC cells were 1.5×10^(7) to 7.0×10^(7) copies/μL for 2 h,the optimal working concentration of the primary and secondary antibody were both1∶100.Using the method established in this experiment,it confirmed that the sera from experimental Mccp-infected goats can block the adhesion of Mccp to GTC.Those results showed that the IFA is able to detect the adhesion of Mccp to host cell,which provide an effective tool for the detection of Mccp infection,identification of adhesion factors and exploring adhesion mechanisms.