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

Thavaselvam, Duraipandian; Kumar, Adarsh; Tiwari, Sapana; Mishra, Manvi; Prakash, Avinash

doi:10.1099/jmm.0.017566-0

Cited by 32 publications

(40 citation statements)

References 30 publications

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“…suis, B. ovis, B. canis, B. neotomae and B. melitensis. Recombinant OMP28 was sensitive and specific for diagnosis of Brucella infection in animals by indirect enzyme-linked immunosorbent assay (I-ELISA) (Kumar et al, 2008;Gupta et al, 2010;Thavaselvam et al, 2010;Liu et al, 2011;Dong-Bao et al, 2012;Lim et al, 2012;Qiu et al, 2012;Azizpour et al, 2013;Kim et al, 2013;Xin et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and immunochemical characterization of recombinant OMP28 protein of Brucella species

Manat¹,

Shustov²,

Evtehova³

et al. 2016

Open Vet J.

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Brucellosis is the lion's share of infectious disease of animals and it has a particular socio-economic importance for the Republic of Kazakhstan. Sixty percent of epizootic outbreaks of brucellosis identified in the Commonwealth of Independent States (CIS) originated from Kazakhstan in recent years. Definitive diagnosis of brucellosis remains a difficult task. Precisely for this reason, we evaluated a purified recombinant out membrane protein 28 (rOMP28) of Brucella species (Brucella spp.) produced in Escherichia coli (E. coli) as a diagnostic antigen in an Indirect ELISA (I-ELISA) for bovine brucellosis. The gene encoding OMP28 was synthesized using a two-round PCR procedure. In order to produce the rOMP28, the de novo synthesized DNA was cloned into the expression vector pET-22b(+). Then, the rOMP28 was expressed in E. coli system and characterized in the present study. We further estimated the diagnostic potential of purified rOMP28 of Brucella spp. for screening bovine sera. To determine if rOMP28 has a valuable benefit for use in the serodiagnosis of bovine brucellosis, rOMP28-based I-ELISA was performed. Brucella spp. positive (n=62) and Brucella spp. negative (n=28) samples from tube agglutination test (TAT) were positive (n=59) and negative (n=27) by I-ELISA, respectively. These findings show that the rOMP28 of Brucella spp. could be a good candidate for improving serological diagnostic methods for bovine brucellosis.

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Section: Introductionmentioning

confidence: 99%

Expression, purification and immunochemical characterization of recombinant OMP28 protein of Brucella species

Manat¹,

Shustov²,

Evtehova³

et al. 2016

Open Vet J.

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“…Omp28 is a conserved protein found in B. melitensis. The high level expression of Omp28 of B. melitensis was previously achieved in an E. coli system, and an ELISA system and a dot-blot format were applied to diagnose human and bovine brucellosis [2,13,18]. However, the expression of Omp28 of B. abortus has not been reported, and serodiagnosis using the rOmp28 of B. abortus in bovine brucellosis has not been studied.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Recombinant 28 kDa Outer Membrane Protein of Brucella abortus for the Clinical Diagnosis of Bovine Brucellosis in Korea

Lim

Kim

Lee

et al. 2012

The Journal of Veterinary Medical Science

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ABATRACT. Brucella spp. are Gram-negative, facultative, intracellular coccobacilli that are pathogenic to a variety of mammals, including ruminants and humans. The conventional serological test for diagnosing brucellosis in cattle in Korea is the standard tube agglutination test. However, agglutination tests sometimes give false-positive results due to cross-reactions with other pathogens. The outer membrane proteins of Brucella species have been extensively studied for their immunogenicity and serodiagnostic applications. However, an application of B. abortus OMPs for serodiagnosis has not been successfully established. In this study, cloning and expression of B. abortus Omp28, a group 3 antigen, were accomplished by PCR amplification cloning into a pMAL expression system, and purification of a recombinant Omp28 (rOmp28). The immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive bovine serum. To determine whether rOmp2 has a potential benefit for use in the serodiagnosis of bovine brucellosis, rOmp28-based ELISA and latex bead agglutination test were performed. B. abortus positive (n=122) or negative (n=88) Microorganisms belonging to the genus Brucella are Gram-negative, facultative, intracellular bacteria of zoonotic importance. Like other intracellular pathogens, Brucella spp. are virulent mainly because of their ability to avoid lysosomal degradation and proliferate within macrophages, leading to the establishment of a chronic infection in the host [6,7,14]. Brucella spp. may have either smooth or rough lipopolysaccharide (S-LPS or R-LPS), depending on the presence or absence of O-polysaccharide chains, respectively. Accordingly, the O-polysaccharide chains of LPS are thought to be necessary for the pathogenicity of Brucella strains bearing S-LPS [1,12,19,23]. The LPS of smooth Brucella species is by far the most immunostimulatory antigen when compared to other antigenic molecules. LPS elicits a long-lasting serological response in both vaccinated and infected animals [1,11,19,22]. These serological tests are mainly based on the detection of antibodies directed against the LPS portion of the bacterial cell membrane. For these reasons, it is difficult to differentiate between vaccinated and infected animals using LPS-based serological diagnosis [16,20]. In addition, serological tests based on anti-LPS antibodies give false positives because of cross-reactivity with other Gram-negative bacteria such as Yersinia enterocolitica O:9, Salmonella spp. and Escherichia coli [1]. Outer membrane proteins (OMPs) of Brucella, a non-LPS group of immunogens, have been the focus of vaccine development and the diagnosis of brucellosis [3,5,10,12]. The OMP antigens (Ags) of Brucella are categorized into three groups (1, 2 and 3) according to their molecular weights. Groups 1, 2 and 3 have approximate molecular masses of 94, 41 to 43, and 25 to 30 kDa, respectively [21]. In this study, the Omp28 coding gene of B. abortus was cloned and expressed using the pMAL expression system, and the rOmp...

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“…The omp28 gene of B. melitensis was cloned in pQE30UA in our previous study, and purified rOmp28 protein was prepared as described earlier (9,11). The clone was inoculated into 10 ml LB medium containing kanamycin (25 g/ml) to prepare a starter culture for the expression and purification of the rOmp28 antigen.…”

Section: Methodsmentioning

confidence: 99%

“…To overcome these problems and to increase the sensitivity and specificity of the test system, this study was designed to use recombinant proteins as antigens in ELISAs. The cloning and expression of the recombinant Omp28 (rOmp28) protein of B. melitensis and screening for its diagnostic potential were reported in our previous study (9). In the present study, we have compared the efficacies of rOmp28 and rOmp31 proteins with those of the cell envelope and whole-cell sonicated antigen by indirect plate ELISAs and also with conventional agglutination tests for the serodiagnosis of human brucellosis.…”

mentioning

confidence: 99%

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Tiwari

Kumar

Thavaselvam

et al. 2013

Clin Vaccine Immunol

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bBrucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis. Brucellosis is one of the world's major zoonoses, caused by bacteria of the genus Brucella. It is a serious public health problem in India and in other developing countries. The transmission of Brucella infection and its prevalence in a region depend upon several factors, like food habits, methods of processing milk and milk products, social customs, husbandry practices, climatic conditions, socioeconomic status, and environment hygiene (1). The disease has been recognized as one of the most common laboratory-acquired infections; it has been reported to occur in clinical research and production laboratories. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations, and its diagnosis requires microbiological confirmations by means of isolation from blood cultures or demonstration of the presence of specific antibodies by serological tests (2). Blood cultures provide definite proof of brucellosis but may not provi...

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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

Cited by 32 publications

References 30 publications

Expression, purification and immunochemical characterization of recombinant OMP28 protein of <i>Brucella</i> species

Expression, purification and immunochemical characterization of recombinant OMP28 protein of <i>Brucella</i> species

Evaluation of Recombinant 28 kDa Outer Membrane Protein of <i>Brucella abortus</i> for the Clinical Diagnosis of Bovine Brucellosis in Korea

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

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