Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine

Soheil Yousefi, Mojtaba Tahmoorespur, Mohammad Hadi Sekhavati

Abstract


Brucellosis is a well-known domestic animal infectious disease, which is caused by Brucella bacterium. The outer membrane protein 25 kDa (Omp25) gene plays an important role in simulating of TNF-α, IFN-α, macrophage, and cytokines cells. In the current study molecular cloning and expression analysis of Omp25 gene for designing a subunit vaccine against Brucella was investigated. Amplifying the full length of candidate gene was performed using specific primers. Sub-cloning of this gene conducted using pTZ57R/T vector in TOP10F`strain of Escherichia coli(E.coli) as the host. Also, pET32(a)+ vector used for expression in BL21 (DE3) strain of E.coli. Omp25 gene with 642 bp size was amplified and cloned successfully. The expression results were confirmed by sequencing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses which showed 42 kDa protein band correctly. Also, phylogenic analysis showed this gene has a near genetic relation with other Brucella strains. According to our results we can propose this gene as a candidate useful for stimulation of cell-mediated and humoral immunity system in future study.


Keywords


Brucella melitensis Rev1; Omp25; Phylogenic analysis; Gene expression

Full Text:

PDF

References


Pappas G, Papadimitriou P, Christou L, Akritidis N. Future trends in human brucellosis treatmen. Expert Opin Investig Drugs. 2006;15(10):1141–1149.

Pappas G. The changing Brucella ecology: novel reservoirs, new threats. Int J Antimicrob Agents. 2010;36 suppl 1:S8-11.

Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis. 2007;7(12):775-786.

Corbel MJ. Brucellosis in humans and animals. Produced by the World Health Organization in

collaboration with the Food and Agriculture Organization of the United Nations and World Organisation for Animal Health, 2006.

Cassataro J, Estein SM, Pasquevich KA, Velikovsky CA, de la Barrera S, Bowden R, et al.Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection. Infect Immun. 2005;73(12):8079-8088.

Seleem MN, Boyle SM, Sriranganthan N. Brucellosis: A re-emerging zoonosis. Vet Microbiol. 2010;140(3-4):392-398.

Golshani M, Rafati S, Jahanian-Najafabadi A, Nejati-Moheimani M, Siadat SD, Shahcheraghi F, et al. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens. Res Pharm Sci. 2015;10(5):436-445.

Gupta VK, Vohra J, Kumari R, Kumaresan G, Vihan VS. Identification of Brucella isolated from goats using PstI sitepolymorphism at Omp2 gene loci. Indian J Anim Sci. 2012;82(3):240–243.

Edmonds MD, Cloeckaert A, Elzer PH. Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis. Vet Microbiol. 2002;88(3):205–221.

Goel D, Bhatnagar R. Intradermal immunization with outer membrane protein 25protects Balb/c mice from virulent B. abortus 544. Mol Immunol. 2012;51(2):159–168.

Jubier-Maurin V, Boigegrain RA, Cloeckaert A, Gross A, Alvarez-Martinez MT, Terraza A, et al. Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages. Infect Immun. 2001;69(8):4823–4830.

Delpino MV, Estein SM, Fossati CA, Baldi PC, Cassataro J. Vaccination with Brucella recombinant DnaK and SurA, proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine. 2007;25(37-38):6721-6729.

Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. (3rd ed). Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY. 2001.

Tahmoorespur M, Sekhavati MH, Yousefi S, Abbassi-Daloii T, Azghandi M, Akbari R. In silico analysis of Omp25 and BLS Brucella melitensis antigens for designing subunit vaccine. Arch Razi Inst. 2016;71(1):35-42.

Cloeckaert A, Vizcaino N, Paquet JY, Bowden RA, Elzer PH. Major outer membrane proteins of Brucella spp.: past, present and future. Vet Microbiol. 2002;90(1-4):229-247.

Goel D, Rajendran V, Ghosh PC, Bhatnagar R. Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544. Vaccine. 2013;31(8):1231–1237.

LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF. McCoy JM. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y). 1993;11(2):187–193.

Cloeckaert A, Verger JM, Grayon M, Zygmunt MS, Grépinet O. Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene. Infect Immun. 1996;64(6):2047-2055.


Refbacks

  • There are currently no refbacks.


Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.