【摘要】 将布鲁氏菌BP26蛋白基因克隆入pET-28a原核表达载体,经表达、纯化后进行Western blot分析。以该蛋白为包被抗原,建立布鲁氏菌BP26抗体iELISA检测方法,并进行特异性、敏感性、符合率等试验。结果表明:成功构建重组原核表达载体pET28a-BP26,表达和纯化后获得目的蛋白浓度为680μg/mL,Western blot显示与牛和羊血清均具有良好的免疫学活性;建立并优化了BP26抗体iELISA检测方法,抗原包被浓度为13.6μg/mL、血清稀释倍数为1∶100、封闭条件为1.5%乳清蛋白、37℃1 h,二抗稀释倍数为1:2000,37°显色15 min;用建立的iELISA检测方法与传统虎红平板凝集试验比较,413份羊血清样品的符合率为92.2%,186份牛血清样品的符合率为94.8%。该方法可大规模应用于临床牛和羊血清样品检测,为布鲁氏菌病抗体监测及净化提供技术支持。更多还原

【Abstract】 The BP26 protein gene of Brucella suis was cloned into the pET-28 a prokaryotic expression vector. The recombinant plasmid was induced by IPTG, purified by affinity chromatography and detected by Western blot. Then the detection of BP26 antibody indirect ELISA was established, and the specificity, sensitivity, coincidence rate were tested. The results showed that the recombinant plasmid pET28 a-BP26 was successfully constructed. The concentration of purification protein was 680 μg/mL, and Western blot showed it kept the antigen activity with both bovine and sheep serum. At the end, the indirect ELISA diagnostic method were established, with the antigen concentration of 13.6 μg/mL, serum dilution ratio 1:100 times, the blocking buffer 1.5% lactalbumin with 1 hour in 37°, secondary antibody dilution ratio 1:2000, color condition 15 min in 37°. Comparing the established iELISA detection method with the traditional Tiger Red plate agglutination test, the coincidence rate of 413 goat serum samples was 92.2%, and the coincidence rate of 186 bovine serum samples was 94.8%. The BP26 antibody indirect ELISA diagnostic method can be used in large scale clinical bovine and sheep samples detection, providing technical support for antibody surveillance and purification of Brucella suis.更多还原