布鲁菌bp26基因的表达与bp26-间接ELISA抗体检测试剂盒的研制

Expression of bp26 Gene of Brucella and Development of bp26-indirect ELISA Diagnostic Kit

为了研制布鲁菌bp26-间接ELISA抗体检测试剂盒,将布鲁菌bp26基因克隆至原核表达载体pET-32a(+),重组质粒转化大肠杆菌BL21(DE3)中诱导表达,以纯化后的重组蛋白bp26包被酶标板,优化ELISA反应条件,组装试剂盒。SDS-PAGE和Western blotting分析结果表明,重组蛋白分子质量约为41ku,经IPTG诱导后高效表达,且具有良好的免疫原性;优化试验确定重组抗原最佳包被浓度为3.6μg/mL,血清样本的阳性临界值为0.370;特异性试验结果表明,重组蛋白与牛结核菌、副猪嗜血杆菌、链球菌等多种病原体的阳性血清均无交叉反应;敏感性试验结果表明,血清稀释至1∶12800时,仍检测为阳性;该试剂盒的批内、批间变异系数均小于10%;用该试剂盒检测241份临床牛血清样本,并与试管凝集试验(SAT)检测结果比较,两者符合率为97.1%。原核表达的布鲁菌bp26具有良好的免疫原性,研制的布鲁菌bp26-间接ELISA抗体检测试剂盒敏感性、特异性和重复性较好,可为布鲁菌基因缺失标记疫苗的应用提供配套的血清学诊断方法。

In order to develop an indirect ELISA method for detecting antibody against Brucella, the bp26 gene of Brucella was inserted into pET-32a（＋） vector and the recombinant plasmid was transformed into E. coli BL21 （DE3） and expressed. Using the purified recombinant protein bp26 as coating antigen to establish indirect ELISA method, and the reaction conditions were optimized and kit was assembled. The recombinant protein was expressed in high level in BL21（DE3） as confirmed by SDS-PAGE, and showed Strong immunological reaction with Brucella positive serum detected by Western blotting. The test confirmed that the best coating concentration of the recombinant antigen was 3.6 μg/mL, and the critical value of positive ser- um samples was 0. 370. The specificity test showed that there were no cross-reactivity between the recombinant protein and TB, HPS, SC and other pathogens positive serum. The sensitivity tests indicated that the diluted serum ratio was 1 ： 12800. The coeffi- cient of variation of intra-assay and inter-assay were less than 10%. There was 97.1% coincidence rate between the indirect ELISA kit and SAT based on detection of 241 clinical bovine serum samples. In conclusion,the prokaryotic expression of recombinant protein bp26 had good immunogenicity, and the established indirect ELISA method had good sensitivity, specificity and reproducibility, which could provide a serological method for distinguishing between bp26 gene-detected marked vaccine immunization and natural infection.