Design and Production of a Novel Recombinant Chimeric IL2-Omp31 Antigen against Brucella Infection

Document Type : Original Articles

Authors

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

Abstract

Brucellosis is a zoonotic disease in human and animals. Brucella melitensis is one of the most pathogenic species of Brucella in goat and sheep. Omp31 is an outer membrane protein of Brucella that acts as an immunogenic protein. Cytokines are glycoproteins with low molecular weight that play the role of an immune adjuvant and regulate immune responses. Interleukin-2 is one of the most important cytokines, which are secreted by the white blood cells and involved in T cell immune responses. In the present study, a chimeric Omp31-Interleukin2 recombinant protein was generated by means of genetic engineering techniques. This chimeric coding sequence was amplified by using specific primers and using Splicing Overlap Extension (SOE) PCR technique. The fusion of the two mentioned proteins was accomplished using a rigid linker. The generated chimeric IL2-Omp31 fragment was TA cloned, and then subcloned into pEt22b vector as an expression vector. The chimeric protein was successfully expressed in E. coli BL21 (DE3) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and also Western-blotting analysis. Finally, in order to assess the antigenic features of the recombinant chimeric IL2-Opm31 protein, its secondary structure and antigenicity were predicted in silico.

Keywords

Main Subjects

Article Title [French]

Conception et Production d'un Nouvel Antigène Chimique IL2-Omp31 Recombinant Contre l'infection à Brucella

Abstract [French]

La brucellose est une maladie zoonotique chez l'homme et les animaux. Brucella melitensis est l'une des espèces les plus pathogènes de Brucella chez les chèvres et les moutons. Omp31 est une protéine de membrane externe de Brucella qui agit comme une protéine immunogène. Les cytokines sont des glycoprotéines de faible poids moléculaire qui jouent le rôle d'un adjuvant immunitaire et régulent les réponses immunitaires. L'interleukine-2 est l'une des cytokines les plus importantes, sécrétées par les globules blancs et impliquées dans les réponses immunitaires des cellules T. Dans la présente étude, une protéine recombinante chimère Omp31-Interleukine2 a été générée au moyen de techniques de génie génétique. Cette séquence de codage chimérique a été amplifiée en utilisant des amorces spécifiques et en utilisant la technique de PCR par Extension de Chevauchement d'Épissage" (ECE). La fusion des deux protéines mentionnées a été réalisée en utilisant un connecteur rigide. Le fragment chimérique d'IL2-Omp31 généré a été TA cloné, puis sous-cloné dans le vecteur pEt22b en tant que vecteur d'expression. La protéine chimère a été exprimée avec succès dans E. coli BL21 (DE3) et confirmée par électrophorèse sur gel de dodécylsulfate de sodium polyacrylamide (SDS-PAGE) et également par analyse par transfert Western. Enfin, afin d'évaluer les caractéristiques antigéniques de la protéine chimérique IL2-Opm31 recombinante, sa structure secondaire et son antigénicité ont été prédites in silico.

Keywords [French]

  • Brucella melitensis
  • Interleukine-2
  • Omp31
  • Cytokines

References

Abbassi‐Daloii, T., Yousefi, S., Sekhavati, M.H. and Tahmoorespur, M., 2018. Impact of heat shock protein 60 KD in combination with outer membrane proteins on immune response against Brucella melitensis. Apmis, 126(1), pp.65-75.
Arai, R., Ueda, H., Kitayama, A., Kamiya, N., Nagamune, T., 2001. Design of the linkers which effectively separate domains of a bifunctional fusion protein. J Protein Engin 14, 529-532.
Avila-Calderon, E.D., Merino, A.L., Sriranganathan, N., Boyle, S.M., Contreras-Rodríguez, A., 2013. A History of the Development of Brucella Vaccines. J BioMed Research Int 2013, Article ID 743509
Cloeckaert, A., MaggyGrayon, B., Olivier Grépinet, B., 2002. Identification of Brucellamelitensis vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene. J Vaccine. 20, 2546–2550.
Bowden, R.A., Cloeckaert, A., Zygmunt, M.S., Dubray, G., 1995. Outer-membrane protein and rough lipopolysaccharide-specific monoclonal antibodies protect mice against Brucellaovis. J Med Microbiol 43, 344-347.
Bruce D., 1887. Note on the discovery of a microorganism inMalta fever. J Practitioner 39, 161–170.
Chen,X., Zaro, J., Shen, W.C., 2013. Fusion Protein Linkers: Property, Design and Functionalit. J. NIH-PA 65, 1357–1369.
Chen, X., Zaro, J.L., Shen, W.C., 2012. Fusion protein linkers: Property, design and functionality. J Adv Drug Deliv Rev 65, 1357-1369.
Cloeckaert, A., Verger, J.M., Grayon, M., Paquet, J.Y., Garin-Bastuji, B., Foster, G., Godfroid, J., 2001. Classification of Brucella spp. isolated from marine mammals by DNA polymor-phism at the Omp2 locus. J Microbes Infect 3, 729–738.
Cloeckaert, A., Vizcaino, N., Paquet, J.Y., Bowden, R.A., Elzer, P.H., 2002. Major outer membrane proteins of Brucella spp. past, present and future. J Vet Microbiol 90, 229-247.
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 Brucellaabortus infection in BALB/c mice. J Vaccine 25, 6721-6729.
Fowell, D., McKnight, A.J., Powrie, F., Dyke, R., Mason, D., 1991. Subsets of CD4+ T cells and their role in the induction and prevention of autoimmunity. J Immunol Rev 123,37–64.
Galinska, E.M., Zagorski, J., 2013. Brucellosis in humans – etiology, diagnostics, clinicalforms. J Ann Agric Environ Med 20,233–238.
Geourjon, C., Deléage, G., 1995. SOPMA. Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. J Comput Appl Biosci 11, 681-684.
Ghasemi, A., Jeddi-Tehrani, M., Mautner, J., Salari, M.H., Zarnani, A.H.,2015. 'Simultaneous immunization of mice with Omp31 and TF provides protection against Brucellamelitensis infection'. J Vaccine1333, 5532-5538.
Golshani, M., Zandi, P., Bouzari, S., 2014. In silico Design of Truncated Omp31 Protein of Brucellamelitensis: Its Cloning and High Level Expression in Escherichia coli. J Vaccine Res 1, 16-21.
Gwida, M., Al Dahouk, S., Melzer, F., Rosler, U., Neubauer, H., Tomaso, H., 2010. Brucellosis– regionally emerging zoonotic disease? J Croat Med 51,289–295.
Haag, A.F., Myka, K.K., Arnold, M.F., Caro-Hernandez, P., Ferguson, G.P., 2010. Importance of lipopolysaccharide and cyclic beta-1,2-glucans in Brucella-mammalian infections. J Microbiol 124509
Kittelberger, R., Hilbink, F., Hansen, M.F., Ross, G.P., de Lisle, G.W., Cloeckaert, A., de Bruyn, J., 1995. Identification and characterization of immunodominant antigens during the course of infection with Brucellaovis. J Vet Diagn Invest 7, 210-218.
Lee, M., Bang, K., Kwon, H., Cho, S.,2013.Enhanced antibacterial activity of an attacincoleoptericin hybrid protein fused with a helical linker. J Molecul Biol 40, 3953-3960.
Leonard, W.J., 2001. Cytokines and immunodeficiency diseases. J Nat Rev Immunol 1, 200–208.
Lin, Y., Ya-Li, Q., Wei-Cheng, B., Xiao-Lan, Y., Hong-Liang, H., Huan-Chun, C., 2004. Expression of an interleukin-6 - interleukin-2 fusion protein (pIL-6-IL-2) in P. pastoris. J Cytokine Netw 15, 240–246.
Mossman, T.R., Coffman, R.L., 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. J Annu Rev Immunol 7,145–73.
Olsen, S.C., 2013. Recent developments in livestock and wildlife brucellosis vaccination. J Rev Sci Tech 32, 207-217.
Pappas, G., Akritidis, N., Bosilkovski, M., Tsianos, E., 2005. Brucellosis. J Engl Med 352, 2325–2336.
Ping, L., Ming-Guang, F., 2008. Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Microbiol Biotechnol 79, 579–587.
Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, in Cold SpringHarbor Laboratory Press, pp. 203-206.
Schijns, V.E., 2000. Immunological concepts ofvaccine adjuvant activity. J Curr Opin Immunol 12, 456–463.
Sekhavati, M.H., Tahmoorespur, M., Abbassi-Daloii, T., Yousefi, S., Khabiri, A.A., Akbari, R., Shekari, E., 2015. Dual Promoter Vector Construction for Simultaneous Gene Expression Using SOE-PCR Technique. Iran J Appl Anim Sci 5, 491-495.
Vahedi, F., Talebi, A.F., Ghorbani, E., Behroozikhah, A.M., Shahriari-Ahmadi, F., Mahmoudi, M., 2011. Isolation, cloning and expression of the Brucellamelitensis Omp31 gene. J Vet Res Shiraz Uni 12, 156-162.
Vizcaino, N., Cloeckaert, A., Zygmunt, M.S., Dubray, G., 1996. Cloning, nucleotide sequence, and expression of the Brucellamelitensis omp31 gene coding for an immunogenic major outer membrane protein. J Infect Immun.64, 3744–3751.
Wales, G.R., Baird, M.A., Davies, N.M., Buchan, G.S., 2005. Fusing subunit antigens to interleukin-2 and encapsulating them in liposomes improves their antigenicity but not their protective efficacy. J Vaccine 23, 2339–2341.
Wass, M.N., Sternberg, M.J., 2009. Prediction of ligand binding sites using homologous structures and conservation at CASP8. J Proteins 77, 147-151.
 Yang, X., Hudson, M., Walters, N., Bargatze, R.F., Pascual, D.W., 2005. Selection of pro-tective epitopes for Brucellamelitensis by DNA vaccination. J Infect Immun 7297–7303.
Yousefi, S., Tahmoorespur, M., Sekhavati, M.H., 2016. Cloning, expression and molecular analysis of Iranian Brucellamelitensis Omp25 gene for designing a subunit vaccine. J Res Pharma Sci 11, 405-411.
Yousefi, S., Abbassi-Daloii, T., Sekhavati, M.H. and Tahmoorespur, M., 2018. Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen. Microbial pathogenesis, 115, pp.50-56.
Zhang, H., Qiu, Y., Zhao, Y., Liu, X., Liu, M., Yu, A., 2014. Immunogenicity of oral vaccination with Lactococcuslactis derived vaccine candidate antigen (UreB) of Helicobacter pylori fused with the human interleukin2 as adjuvant. J Molecular Cell Prob 28, 25-30.
Zhang, H.Y., Sun, S.H., Guo, Y.J., Zhu, W.J., Shi, K., Xu, G.X., Wang, J., 2008. Optimization strategy for plasmid DNAs containing multiple-epitopes of foot-and-mouth diseasevirus by cis-expression with IL-2. J Vaccine 26, 769-777.
Archives of Razi Institute
Volume 73, Issue 3
September 2018
Pages 199-206

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