Abstract: 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 serum 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 coefficient 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.

Key words: Brucella; prokaryotic expression; indirect ELISA