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

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.