Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis

Abstract

BackgroundBrucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay).Method Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23).ResultThe rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student’s t test was performed on the data.ConclusionOmp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.

Brucellosis detection and the role of Brucella spp. cell wall proteins

“…In addition, blood sera from patients with other febrile diseases and healthy donors were used as controls. The sensitivity and specificity of i-ELISA/rOmp2a and western blotting were very high (94%–96%) [ 32 ]. Sufficient specificity was also established for rOmp2b: sera from mice inoculated with E. coli O:157 , Salmonella enterica , and Y. enterocolitica O:9 did not react significantly with rOmp2b in an i-ELISA.…”

3D structure of a Brucella melitensis porin: molecular modelling in lipid membranes

“…Brucella melitensis has a high pathogenicity and this work focus on one of its major OMP: Omp2a, a 39 kDa nonspecific trimeric porin (DelVecchio et al, 2002) that was recently shown to be a potential antigen candidate for serodiagnosis of human brucellosis (Pathak, 2017). Outer membrane (OM) permeability assays indicated that Omp2a increases the hydrophilic permeability of E. coli, consistent with pore-forming activity (Marquis & Ficht, 1993).…”