副结核分支杆菌Hed蛋白的抗原性分析及在间接ELISA中的应用

Antigenicity analysis of the Mycobacterium paratuberculosis Hed protein and its application in indirect ELISA

从副结核分支杆菌K-10菌株克隆出840 bp的hed基因片段,插入原核表达载体pET-32a(+),转化至Escherichia coliBL21(DE3),在0.8 mmol.L-1 IPTG诱导下进行表达,纯化重组蛋白,将其进行Western-blot分析、小鼠免疫试验,包被ELISA板,建立间接ELISA方法。结果显示:重组蛋白具有良好的抗原性,方阵滴定法确定了间接ELISA方法的抗血清最佳稀释度(100倍)和抗原包被浓度(4.4μg.mL-1),该方法具有较好的特异性和敏感性。

To detect antigenicity of Hed protein of Mycobacterium paratuberculosis（Map）,and establish the ELISA diagnosis,hed gene was cloned from Mycobacterium paratuberculosis K-10,and inserted to pET-32a（＋）vector.The recombinant plasmid was transformed into competent Escherichia coli BL21（DE3）.The recombinant protein was expressed,purified,and detected by Western-blot and the mice immunologic test.After that it was applied in indirect ELISA.The results showed the recombinant proteins possessed the good antigenicity.The ELISA assay based on Hed protein was more specific and sensitive.