绵羊肺炎支原体P71蛋白分段表达及其间接ELISA方法的建立  

Development of an indirect ELISA method with the main antigentic region of Mycoplasma ovipneumoniae P71 protein expressed in E.coli

作　　者：黄海碧[1] 郑佳琪[1] 王仁超[1] 王晓晖[1] 陈德浩[1] 王灵芮 郭逸贤 郝永清[1] 

机构地区：[1]内蒙古农业大学兽医学院/农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《中国预防兽医学报》2015年第8期623-627,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2011BAD18B01;2012BAD13B00)

摘　　要：为研究绵羊肺炎支原体(MO)P71蛋白免疫学相关功能及建立MO的快速检测方法,本研究采用PCR扩增MO P71基因片段,测序后利用生物学软件分析P71蛋白的抗原表位,选取抗原表位富集的3个基因片段P71-1(1 bp^516 bp)、P71-2(505 bp^1 338 bp)和P71-3(1 327 bp^1 905 bp)分别克隆至原核表达载体p ET-32a中进行表达。分别利用western blot和间接ELISA方法对表达产物的免疫原性、反应原性进行鉴定,以筛选的优势抗原蛋白为诊断抗原,通过对反应条件的优化,建立检测MO抗体的间接ELISA方法。结果表明:重组蛋白P71-3产生的抗体效价最高,与MO阳性血清的结合能力最强,阴阳性临界值为:OD450nm值=0.292。该方法与口蹄疫、小反刍兽疫、副结核、羊布鲁菌病、结核阳性血清均无交叉反应,具有极强的特异性。组内组间变异系数均小于10%,重复性较好。利用该方法与间接血凝试验对150份临床血清样品进行检测,结果显示,两者符合率为96.67%。本研究为进一步研制MO ELISA检测试剂盒奠定了基础。To identify the different antigenic regions of the protein P71 of Mycoplasma ovipneumoniae and establish a rapid method for detection of antibody against M. ovipneumoniae, three encoding immunodominant regions of M. ovipneumoniae P71-1 （1 bp-516 bp）, P71-2（505 bp-1,338 bp） and P71-3 （1,327 bp-1,905 bp） were cloned into pET-32a for expressing in E.coli. Then, the expressed recombinant protein of P71-3 （rP71-3） had a highest affinity to antibody against M.ovipneumoniae than that of others by indirect ELISA detection, which was used as coating antigen for development of the indirect ELISA to detect M.ovipneurnoniae. The results showed the assay were specific to detect the antibody against M.ovipneumoniae with cut-off value of 0.292, but had no cross-reaction with other antisera. This study provided a basis for the development of the commercial ELISA kit for detection of antibody against M. ovipneumoniae.

关 键 词：绵羊肺炎支原体 黏附基因 表达 抗原性分析 ELISA 

分 类 号：S852.62[农业科学—基础兽医学]