Development Of An IELISA For Detection And Application Of Brucella Abortous VirB5

Brucellosis is a zoonotic disease which is caused by Brucella. It’s characterized by reproductive organs and fetal membrane inflammation, cause abortion, infertility and various local lesions of the organization. The disease bring serious damage to animal husbandry and human health. In order to establish a rapid and accurate ELISA method for detection of brucellosis antibody, in this research, 4 serological tests for bovine brucellosis detection were compared and analyzed. The coincidence rate of RBPT and ELISA, SAT and CFT was more than 95% as well as the Kappa values > 0.75. Take CFT as a standard, the sensitivity of RBPT and ELISA is better, but existing a certain false positive. The specificity of SAT is better, but existing a certain false negative. ELISA has better sensitively and specificity than RBPT and SAT. We choose Brucella VirB5 as a candidate gene, and daownloaded this gene from GeneBank, one pair of specific primers to VirB5 was designed and amplified from the genomic DNA template(A19) of B.abortus by a polymerase chain reaction(PCR) technique. The VirB5 gene was cloned into the prokaryotic expression vectorpET-32a(+), after sequencing following PCR, it was confirmed that the expression vector pET-32a(+)-VirB5 was constructed successfully. The recombinant plasmid was transformed into Escherichia coli BL21 cells(processed by DE3). It was suecessuflly expressed as a fusion poretin with glutathion transferase protein at the presence of IPTG and was identified by SDS-PAGE and Western blotting. The purification of pET-32a(+)-VirB5 protein was coated as a antigen and detected by ELISA, after optimum the coating concentration,the blocking solution and closing time, the dilution titer of secondary antibody and the threshold of positive and negative serum,an indirect ELISA for brucellosis was estabilished, 294 bovine serum was detected using this method、RBPT and commecial IDEXX ELISA kits. The experimental results are as follows: The indirect ELISA for brucellosis was estabilished compared with RBPT sensitivity, specificity, and coincidence rate of 91.30%, 89.60% and 90.14% respectively, IDEXX cattle brucella indirect ELISA kit is sensitivity, specificity, and coincidence rate 95.45%, 94.17% and 94.55%. To sum up, these studies show that ELISA is suitable for screening, CFT for definite diagnosing in clinical detection. It is better to choose more than two tests for diagnosis of bovine brucellosis. Our laboratory established an indirect ELISA based on recombinant proteins VirB5 has good sensitivity, specificity and coincidence; it can be used to detect brucellosis antibodies in clinical detection.