布鲁氏菌VirB12蛋白纯化及其抗体ELISA方法的初步建立

Purification of VirB12 Protein of Brucella and Preliminary Establishment of ELISA for its Antibody

为获得高纯度布鲁氏菌VirB12蛋白,对VirB12分泌表达蛋白先后进行亲和纯化和离子交换纯化。将其作为抗原包被ELISA板,优化反应条件后初步建立了布鲁氏菌抗体的ELISA检测方法。结果表明:蛋白纯化效果良好,重组蛋白纯度可达到98%以上。在建立的ELISA中,纯化重组蛋白的最佳包被浓度为1μg·mL-1,最适封闭剂为5%脱脂奶粉,血清最佳稀释浓度为1∶25,酶标二抗最佳工作浓度为1∶1500。对20份阳性血清及12份阴性血清的检测结果表明,其与SUANOVIR Brucella-Ab C-ELISA试剂盒的总体符合率达到90.6%,表明本试验建立的以VirB12重组蛋白为抗原的ELISA方法可以检测布氏杆菌感染抗体,有望研制成试剂盒用于养殖场布病的检测和净化。

To obtain high purity VirB12 protein of Brucella,affinity purification and ion-exchange purification were conducted.The purified recombinant VirB12 protein was used for coating antigen to establish the ELISA method for the detection of Brucella antibodiesin after optimizing the reaction conditions.The results showed that the purity of the recombined protein could reach more than 98% after purification.In the established ELISA,the optimum coating concentration of purified protein was 1 μg·mL^-1,the optimum sealing solution was 5% skimmed milk powder,the optimum dilution of serum was 1∶25 and the optimum working dilution of enzyme-labeled secondary antibody was 1∶1500.Detection results of 20 positive sera and 12 negative sera indicated that the coincidence rate between the established ELISA method and SUANOVIR Brucella-Ab C-ELISA kit was 94.7%.The results showed that the ELISA method using VirB12 protein as antigen can detect Brucella antibody and be applied to the detection of brucellosis in farms.