Production of Brucella lumazine Synthase Recombinant Protein to Design a Subunit Vaccine against Undulant Fever

Document Type : Original Articles

Authors

1 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Brucella bacterium causes Brucellosis, an infectious disease spreading from animals to human. Brucella lumazine synthase (BLS) is a highly immunogenic protein with adjuvant properties, which has been introduced as an effective protein carrier for vaccine development. This protein also plays a significant role in inducing immune system. This study aimed to clone, express, and purify the BLS gene from Brucella melitensis Rev1. The BLS gene was amplified by particular primers with the restriction enzyme sites as a linker and it was inserted into pTZ57R/T vector. Subsequently, it was ligated into pET32(a)+ expression vector. Recombinant expression vector containing coding sequence of BLS was transformed into E. coli BL21 (DE3) host gene expression and stimulated by 0.1mM IPTG. The results of sequencing showed that there were not any mutations in BLS encoding sequence. The expression results were set by sequencing and endorsed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and western blotting that showed 35 kDa protein band appropriately.

Keywords

Main Subjects

Article Title [French]

Production de la Protéine Recombinante Brucella lumazine Synthase Afin de Concevoir un Vaccin Sous-unitaire contre la Fièvre Ondulante

Abstract [French]

La bactérie Brucella est à l'origine de la Brucellose, une maladie infectieuse se transmettant de l'animal à l'homme. La Brucella lumazine synthase (BLS) est une protéine hautement immunogène dotée de propriétés adjuvantes, qui a été présentée comme un vecteur efficace de transport de protéines pour le développement de vaccins. Cette protéine joue également un rôle important dans l'induction d’une réponse immunitaire. Cette étude visait à cloner, exprimer et purifier le gène BLS de Brucella melitensis Rev1. Le gène BLS a été amplifié par des amorces spécifiques avec les sites d'enzyme de restriction servant de liant afin d’etre inséré dans le vecteur pTZ57R / T. Ensuite, il a été ligaturé dans le vecteur d'expression pET32(a)+. Le vecteur d'expression recombinant contenant la séquence codante de BLS a été transformé dans le système d’expression génique E. coli BL21 (DE3) et stimulé par 0.1 mM d'IPTG. Les résultats du séquençage ont montré qu'il n'y avait pas de mutations dans la séquence codant pour BLS. L'expression de la protéine recombinante a été vérifiée par séquençage ainsi qu’à l'aide d'analyses électrophorèse sur gel de dodécyl sulfate de sodium-polyacrylamide (SDS-PAGE) et d'un transfert de type western montrant une bande protéique de 35 kDa de manière appropriée.

Keywords [French]

  • Brucella melitensis Rev1
  • BLS
  • Expression génique
  • Protéine recombinante

References

Azimi, L., Khoramabadi, N., Mohabati  Mobarez, A.,  Aslz, E., Harzandi, N., R., M., 2012. Survey of protection of recombinant cell surface protein 31kDa from Brucella melitensis in BALB/c mice. Journal of Pure and Applied Microbiology 6, 69-73.
Cassataro, J., Estein, S.M., Pasquevich, K.A., Velikovsky, C.A., de la Barrera, S., Bowden, R., Fossati, C.A., Giambartolomei, G.H., 2005. Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ Thelper 1 response that protects against Brucella melitensis infection. Infect Immun 73, 8079-8088.
Cassataro, J., Pasquevich, K.A., Estein, S.M., Laplagne, D.A., Zwerdling, A., de la Barrera, S., 2007. A DNA vaccine coding for the chimera BLSOmp31 induced a better degree of protection against B. ovis and a similar degree of protection against B. melitensis than Rev. 1 vaccination. Vaccine 25, 5958–67.
Clausse, M., Díaz, A.G., Ghersi, G., Zylberman, V., Cassataro, J., Giambartolomei, G.H., 2013. The vaccine candidate BLSOmp31 protects mice against Brucella canis infection. Vaccine 9, 6129-35.
Corbel, M. J., 2006. Brucellosis in Humans and Animals. World Health Organization.
Delpino, M.V., Estein, S.M., Fossati, C.A., Baldi, P.C., Cassataro, J., 2007. Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine 25, 6721-6729.
Diaz Aparicio, E., 2013. Epidemiology of brucellosis in domestic animal caused by Brucella melitensis, Brucela suis and Brucella abortus. Rev Sci Tech Oie 32, 43-50.
Estein, S.M., Fiorentino, M.A., Paolicchi, F.A., Clausse, M., Manazza, J., Cassataro, J., 2009. The polymeric antigen BLSOmp31 confers protection against Brucella Ovis infection in rams. Vaccine 12, 6704-11.
Franco, M.P., Mulder, M., Gilman, R.H., Smits, H.L., 2007. Human brucellosis. Lancet Infect Dis 7, 775-786.
Goldbaum, F.A., Leoni, J., Wallach, J.C., Fossati, C.A., 1993. Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.  J Clin Microbiol Infect Dis 31, 2141–2145.
Ghasemi, A., Salari, M.H., Zarnani, A.H., Pourmand, M.R., Ahmadi, H., Mirshafiey, A., Jeddi-Tehrani, M., 2013. Immune reactivity of Brucella melitensis-vaccinated rabbit serum with recombinant Omp31 and DnaK proteins. Iranian journal of microbiology 5, 19-23.
Hemmen, F., Weynants, Y., Scarcez, T., Letesson, J.J., Saman, E., 1995. Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes. Clin Diagn Lab Immun 2, 263-267.
Mejias, M.P., Ghersi, G., Craig, P.O., Panek, C.A., Bentancor, L.V., Baschkier, A., 2013. Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and Brucella lumazine synthase confers total protection against Shiga toxins in mice. J Immunol 191, 2403–2411.
Pappas, G., Papadimitriou, P., Christou, L., Akritidis, N., 2006. Future trends in human brucellosis treatment. Expert Opin Investig Drugs 15, 1141-1149.
Rosas, G., Fragoso, G., Ainciart, N., Esquivel-Guadarrama, F., Santana, A., Bobes, R.J., 2006. Brucella spp. lumazine synthase: a novel adjuvant and antigen delivery system to effectively induce oral immunity. Infect Immun 8, 1277–86.
Rossi, A.H., Farias, A., Fernandez, J.E., Bonomi, H.R., Goldbaum, F.A., Berguer, P.M., 2015. Brucella spp. lumazine synthase induces a TLR4-mediated protective response against B16 melanoma in mice. PLoS ONE
10:e0126827. 10.1371.
Sambrook, J., Russell, D.W., 2001.  Molecular Cloning:  A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sciutto, A., Toledo, C., Cruz, G., Rosas, G., Meneses, D., Laplagne, N., 2005. Brucella spp. lumazine synthase: A novel antigen delivery system. Vaccine 23, 2784-2790.
Sekhavati, M.H., Majidzadeh Heravi, R., Tahmoorespur, M., Yousefi, S., Abbassi-Daloii, T., Akbari, R., 2015. Cloning, molecular analysis and epitopics prediction of a new chaperone GroEL Brucella melitensis antigen. Iran J Basic Med Sci 18, 499-505.
Tahmoorespur, M., Sekhavati, M.H., Yousefi, S., Abbasi-Daloii, T., Azghandi, M., Akbari, R., 2016. In silico analysis of Omp25 and BLS Brucella melitensis antigens for designing subunit vaccine. Arch Razi Ins 71, 35-42.
Vahedi, F., Talebi, A.F., Ghorbani, E., Behroozikhah, A.M., Shahriari Ahmadi, F., Mahmoudi, M., 2011. Isolation, cloning and expression of the Brucella melitensis Omp31 gene. Iran J Vet Res 12, 156-162.
Velikovsky, C.A., Goldbaum, F.A., Cassataro, J., Estein, S., Bowden, R.A., Bruno, L., 2003. Brucella lumazine synthase elicits a mixed Th1-Th2immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used. Infect Immun 71, 5750–5.
Yang, X., Jerod Aberg, S., Cao, L., Clapp, B., Thornburg, T., 2013. Progress in Brucella vaccine development. Front Biol 8, 60 –77.
Yousefi, S., Tahmoorespur, M., Sekhavati, M.H., 2016. Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine. Res Pharm Sci 11, 405-411.
Yousefi, S., Sekhavati, M.H., Tahmoorespur, M., Abbasi-Daloii, T., 2016. Cloning and characterization of Omp31 Brucella melitensis antigen for designing subunit vaccine. Arch Razi Ins 71, 117-124.
Zhao, Z., Li, M., Luo, D., Xing, L., Wu, S., 2009. Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella. Vaccine 27, 5214–5219.
Archives of Razi Institute
Volume 74, Issue 1
March 2019
Pages 1-6

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