关键词: 布鲁菌：eryA基因, 原核表达, 间接ELISA

Abstract: In this study, the prokaryotic expression of Brucella dominant antigen eryA gene was used to obtain the target protein. On this basis, an indirect ELISA method using this protein as the coating antigen was established. The prokaryotic expression vector pET-30a-eryA was constructed and transformed into E. coli BL21 （DE3） to induce expression with IPTG. The indirect ELISA method was established after the eryA recombinant protein was purified by Ni column affinity, identified and purified by SDS-PAGE, and detected by Western-Blotting on reactogenicity. After optimizing the reaction conditions, the antigen coating concentration was 0.312 5 μg/mL, which was coated at 37 ℃ for 2 h. The blocking solution was 5% gelatin from porcine skin, which was reacted at 37 ℃ for 2 h. The dilution of serum was 1∶100, which was reacted at 37 ℃ for 1 h. The antibody dilution was 1∶8 000, and the reaction was performed at 37 ℃ for 45 min. When the serum dilution was 1∶1 600, and the test was still positive, which proved that the method was sensitive. The specificity of other strains and viruses was negative, which proved that the specificity of indirect ELISA was good. After repetitive test, the coefficients of variation between and within batches were both less than 10%, which proved that the repeatability was good. The total coincidence rate between the established method and the tube agglutination test in detection of clinical serum samples was 95.7%, which proved that the method could be preliminarily applied to clinical diagnosis.

Key words: Brucella, eryA gene, prokaryotic expression, indirect ELISA