Cloning and expression of the immunoreactive Brucella melitensis 28 kDa outer-membrane protein (Omp28) encoding geneand evaluation of the potential of Omp28 for clinical diagnosis of brucellosis

Abstract

Brucellosis is a disease caused by Gram-negative, facultative, intracellularbacteria belonging to the genusBrucella. It is an emerging zoonosis,and an economically important infection of humans and livestock with a worldwidedistribution. Human infection is known to occur through consumption of infectedraw milk, milk products and undercooked or raw meat. Serodiagnosis of brucellosisis carried out by detection of antibodies generated against LPS or whole-cellbacterial extracts by ELISA or agglutination tests using colorimetry. Thepresent study was designed to develop a highly sensitive and specific indirectELISA in both a microtitre plate and dot-blot format employing the recombinantouter-membrane protein 28 (rOmp28). Cloning and expression ofBrucella melitensisOmp28 protein, which is a group 3 antigen, was accomplishedby PCR amplification and cloning of the gene in a pET-28a expression system,followed by Ni-NTA affinity chromatography purification of the His-taggedrecombinant protein. An indirect ELISA in both a microtitre plate and dot-blotformat was optimized with sera collected from three groups: culture-confirmedcases, clinically suspected cases and healthy individuals. The rOmp28 proteinreacted only with the culture-confirmed positive samples and no reaction wasobserved with culture-negative samples, confirming the immunoreactivity ofthe recombinant protein. The test in both formats had a correlation of approximately90 % with the Rose Bengal plate agglutination test (RBPT)and a standard tube agglutination test, assays that are routinely performedfor the serodiagnosis of brucellosis. The sensitivity and specificity of theassay in the plate format were 97.50 and 85.59 %, and in thedot-blot format were 82.05 and 92.43%, respectively, in comparisonwith RBPT. The specificity of this assay was further confirmed by testingsamples that were positive for malaria and typhoid, which gave negative results.This ELISA system in microtitre plates and a dot-blot format will be usefulfor the rapid screening of large numbers of samples for the diagnosis of humanbrucellosis in endemic areas.