In silico cloning and bioinformatics study of Brucella melitensis Omp31 antigen in different mammalian expression vectors

Document Type : Original Research Article (Regular Paper)


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

2 Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.


Brucella melitensis, as a pathogenic gram-negative intracellular bacterium, causes brucellosis in animals and humans. According to literature, the B. melitensis outer membrane protein 31 (Omp31) is considered as an important vaccine candidate against brucellosis. The aim of the current study was to compare three different expression constructs containing B. melitensis Omp31 antigen using bioinformatics analysis approaches to facilitate choosing the best immunogenic construct. The coding sequence of Omp31 gene was PCR amplified, TA cloned and sequenced. The obtained DNA sequence was in silico cloned in pcDNA3.1/Hygro (+), pcDNA3.1/His A and pSecTag2/Hygro mammalian expression vectors using CLC Main Workbench 5.5 software. The Omp31 gene was successfully cloned into the pTZ57R/T vector, and recombination was confirmed by colony PCR and sequencing. Comparison of the obtained Omp31 sequence with other Omp31 gene sequences showed high similarities. Bioinformatics analysis of three different mammalian expression vectors harboring Omp31 gene made it possible to choose the best immunogenic structure for further studies in order to design effective DNA vaccines against brucellosis.


Main Subjects

  • Bachmair, A., 1986. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179-86.
  • Bowden, R.A., Cloeckaert, A., Zygmunt, M.S., Bernard, S., Dubray, G., 1995. Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry. Infection and Immunity 63, 3945-3952.
  • 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+ T helper 1 response that protects against Brucella melitensis infection. Infection and Immunity 73, 8079-8088.
  • Cassataro, J., Pasquevich, K.A., Estein, S.M., Laplagne, D.A., Velikovsky, C.A., de la Barrera, S., Bowden, R., Fossati, C.A., Giambartolomei, G.H., Goldbaum, F.A., 2007. A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev. 1 vaccination. Vaccine 25, 4437-4446.
  • Chen, P., Rayner, S., Hu, K.-h., 2011. Advances of bioinformatics tools applied in virus epitopes prediction. Virologica Sinica 26, 1-7.
  • Cloeckaert, A., Verger, J.M., Grayon, M., Grepinet, O., 1995. Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella. Microbiology 141, 2111-2121.
  • Cloeckaert, A., Verger, J.M., Grayon, M., Zygmunt, M.S., Grepinet, O., 1996. 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. Infection and Immunity 64, 2047-2055.
  • Cloeckaert, A., Vizcaı́no, N., Paquet, J.-Y., Bowden, R.A., Elzer, P.H., 2002. Major outer membrane proteins of Brucella spp.: past, present and future. Veterinary Microbiology 90, 229-247.
  • Dhama, K., Mahendran, M., Gupta, P., Rai, A., 2008. DNA vaccines and their applications in veterinary practice: current perspectives. Veterinary Research Communications 32, 341-356.
  • Dubray, G., Bezard, G., 1980. Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brucellosis. Annals of Veterinary Reseach 11, 367-373.
  • Geourjon, C., Deleage, G., 1995. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Computer Applications in the Biosciences 11, 681-684.
  • Golshani, M., Zandi, P., Bouzari, S., 2014. In silico design of truncated Omp31 protein of Brucella melitensis: Its cloning and high level expression in Escherichia coli. Vaccine Research 1, 16-20.
  • Habtamu, T., Rathore, R., Dhama, K., Karthik, K., 2013. Cloning and molecular characterization of omp31 gene of the Indian isolate of Brucella melitensis. Research Opinions in Animal & Veterinary Sciences 3, 235-243.
  • He, Y., Xiang, Z., 2010. Bioinformatics analysis of Brucella vaccines and vaccine targets using VIOLIN. Immunome Research 6, 1-12.
  • Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132.
  • Larsen, J.E.P., Lund, O., Nielsen, M., 2006. Improved methods for predicting linear B-cell epitopes. Immunome Research 2, 7580-7582.
  • Sambrook, J., Fritsch, E., Maniatis, T., 1989. Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • Vahedi, F., Talebi, A., Ghorbani, E., Behroozikhah, A., Shahriari Ahmadi, F., Mahmoudi, M., 2011. Isolation, cloning and expression of the Brucella melitensis Omp31 gene. Iranian Journal of Veterinary Research 12, 156-162.
  • Wang, P., Sidney, J., Dow, C., Mothe, B., Sette, A., Peters, B., 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Computational Biology 4, e1000048.
  • Wang, P., Sidney, J., Kim, Y., Sette, A., Lund, O., Nielsen, M., Peters, B., 2010. Peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics 11, 1-12.
  • Yang, X., Hudson, M., Walters, N., Bargatze, R.F., Pascual, D.W., 2005. Selection of protective epitopes for Brucella melitensis by DNA vaccination. Infection and Immunity 73, 7297-7303.
  • Yang, X., Skyberg, J.A., Cao, L., Clapp, B., Thornburg, T., Pascual, D.W., 2013. Progress in Brucella vaccine development. Frontiers in Biology 8, 60-77.
  • Yang, Y., Wang, L., Yin, J., Wang, X., Cheng, S., Lang, X., Wang, X., Qu, H., Sun, C., Wang, J., 2011. Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine. Molecular Immunology 49, 175-184.
  • Zhang, W., Liu, J., Zhao, M., Li, Q., 2012. Predicting linear B-cell epitopes by using sequence-derived structural and physicochemical features. International Journal of Data Mining and Bioinformatics 6, 557-569.