边缘无浆体MSP5重组抗原间接ELISA检测方法的建立  

Development of an indirect ELISA based on recombinant MSP5 antigen of Anaplasma marginale

作　　者：马米玲[1] 殷宏[1] 关贵全[1] 李有全[1] 高金亮[1] 刘志杰[1] 刘爱红[1] 刘军龙[1] 任巧云[1] 罗建勋[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室

出　　处：《中国兽医科学》2008年第8期641-645,共5页Chinese Veterinary Science

基　　金：甘肃省自然科学基金项目(0803RJZA043);家畜疫病病原生物学国家重点实验室项目(SKKVEB2008ZZKT035);甘肃省科技重大专项计划项目(0801NKDA033);国家科技基础条件平台项目(2005DKA21100)

摘　　要：将边缘无浆体MSP5基因在大肠杆菌DH5α中表达,获得45 ku的融合蛋白。Western-blot检测证实该表达蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原,建立了边缘无浆体MSP5的间接ELISA检测方法。该方法对边缘无浆体阴性、阳性血清(各100份)的阳性检出率和阴性符合率分别为100%和98%,与双芽巴贝虫、大巴贝虫、环形泰勒虫、瑟氏泰勒虫、衣原体、血吸虫和羊肝片吸虫的阳性血清无交叉反应,与羊无浆体阳性血清有交叉反应。结果表明,建立的间接ELISA方法具有良好的特异性和敏感性。The MSP5 gene of Anaplasma marginale was subcloned into expression vector pGEX-4T-1 to construct a recombinant expression plasmid pGEX-4T-1-MSPS. After the constructed pGEX-4T-1-MSP5 was transformed into Escherichia coli DHSa,a fusion protein of approximately 45 ku in size was expressed at high level. Western-blot analysis showed that the expressed fusion protein could specifically react with anti-serum against A. marginale. An indirect ELISA for diagnosis of anaplasmosis was developed based on the purified fusion protein. Positive coincidence of 100 known positive sera was 100% and negative coincidence of 100 known negative sera was 98% in the indirect ELISA. There were no cross-reaction with sera against Babesia bigemina, B. major, Theileria annulata, T. sergenti, Chlamydia, Fasciola hepatica and Schistosoma japonicum ,respectively,but there was cross-reaction with sera against A. ovis in the indirect ELISA. This result revealed that the indirect ELISA had good specificity and sensitivity.

关 键 词：边缘无浆体 MSP5基因 间接ELISA 

分 类 号：S852.64[农业科学—基础兽医学] R446.61[农业科学—兽医学]