重组微线体MIC10的牛弓形虫抗体间接ELISA检测方法的建立

Development of an indirect ELISA for detecting Toxoplasma gondii infection in cattle based on prokaryotically expressed MIC10

为建立牛弓形虫抗体间接ELISA检测方法,根据弓形虫MIC10基因序列设计合成引物,应用PCR技术扩增MIC10基因,将其克隆至pET-22b载体,构建重组质粒pET-22b-MIC10,将其转化至E.coli BL21(DE3),经IPTG诱导,SDS-PAGE及Western-blot分析表明,该重组蛋白能与弓形虫阳性血清发生特异性反应。重组蛋白经NiNTA纯化,应用牛弓形虫阳性、阴性血清建立了ELISA检测方法,抗原最佳包被质量浓度为5mg/L,二抗稀释倍数为1∶20 000,封闭液为含5%脱脂奶粉的PBST溶液。该方法重复性好、特异性强、敏感性高,为牛弓形虫流行病学调查奠定基础。

In order to establish an indirect ELISA method for detecting Toxoplasma gondii infection in cattle,the primers were designed and synthesized according to the gene sequences of T.gondii MIC10.The gene was amplified by PCR and cloned into pET-22 bvector to construct pET-22b-MIC10.The recombinant plasmid was transformed into BL21(DE3)for expression,and induced by IPTG.SDS-PAGE and Western-blot analysis showed that the expressed protein had specific reaction to T.gondii-positive sera.After the recombinant protein was purified by Ni-NTA,the ELISA method was developed with bovine T.gondii positive and negative sera.The optimal coating concentration of antigen was 5 mg/L,the optimal dilution of second antibody were 1∶20 000 and PBST solution of 5% non-fat dry milk as a blocking solution.The method had a good repeatability,strong specificity and high reproducibility,which layes the foundation for epidemiological survey of T.gondii infection in cattle.