eISSN: 1644-4124
ISSN: 1426-3912
Central European Journal of Immunology
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1/2018
vol. 43
 
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abstract:
Clinical immunology

Diagnostic potential of Brucella melitensis Rev1 native Omp28 precursor in human brucellosis

Ismail Koyuncu
,
Abdurrahman Kocyigit
,
Ahmet Ozer
,
Sahabettin Selek
,
Adnan Kirmit
,
Hasan Karsen

(Centr Eur J Immunol 2018; 43 (1): 81-89)
Online publish date: 2018/03/30
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Serologic tests for brucellosis aim to detect antibodies produced against membranous lipopolysaccharide of bacteria. Diagnostic use of this method is limited due to false positiveness. This study evaluates an alternative antigen to lipopolysaccharides (LPS), outer membrane 28-precursor-protein, of Brucella melitensis Rev1 for its diagnostic value. Omp28 precursor of B. melitensis Rev1 was cloned, expressed, and purified. 6-His and sumo epitope tags were used to tag the protein at N-termini. Omp28 gene was amplified based on the ORF sequence and cloned into a pETSUMO vector. The recombinant construct was propagated in Escherichia coli One Shot® Mach1™ cells then transformed into E. coli BL21(D3) cells for protein expression. The purified protein was studied in an indirect ELISA for diagnosis of brucellosis. Sera samples from 60 patients were screened by ELISA and the results were compared to Rose Bengal plate test. Recombinant antigen-based iELISA has given a successful outcome with the sensitivity, specificity, positive predictive value, and negative predictive value of 87.8%, 96.2%, 96.6%, and 78.78%, respectively. In conclusion, recombinant production and purification of the immunodominant Omp28 precursor protein has been achieved successfully in a one-step process with efficient yield and can be used for diagnosis of brucellosis in humans.
keywords:

ELISA, brucellosis, purification, recombinant Rev1, SUMO fusion, Omp28

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