Production of MPT-64 recombinant protein from virulent strain of Mycobacterium bovis

Document Type : Full paper (Original article)

Authors

1 Ph.D. Student in Biotechnology, Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

3 TB Department, Razi Vaccine and Serum Research Institute, Karaj, Iran and Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals which has been caused by a rod shaped, acid fast bacterium, called Mycobacterium bovis. The rapid and sensitive detection is a great challenge for TB diagnosis. The virulent strains of Mycobacterium tuberculosis complex (MTBC) have 16 different regions of difference (RD) in their genome which encode some important antigens. The major protein of M. bovis 64 (MPT-64) is one of the main immune-stimulating antigens which are encode by RD-2 region. The aim of the present study was cloning, expression and purification of MPT-64 as a protein antigen of M. bovis in a prokaryotic system for the usage in the future diagnostic studies. In this experimental study, the mpt-64 gene with 687 bp has been proliferated from M. bovis whole genome by polymerase chain reaction (PCR) method. The PCR product has been digested by BamHI and HindIII restriction enzymes and cloned into pQE-30 plasmid. The recombinant protein has been expressed in the Escherichia coli M15 with induction by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed protein was analyzed on SDS-PAGE, and purified with Nitrilotriacetic acid (Ni-NTA) column. Finally, its biological properties were confirmed in Western blotting method using specific antibodies. Data showed the successful cloning of mpt-64 gene (as a 687 bp segment) in expression vector. The MPT-64 recombinant protein was ideally expressed and purified as a 24 kDa protein. The result of this study indicated that MPT-64 recombinant protein (24 kDa) has been successfully expressed and purified in a prokaryotic system, so this protein could be used for differential diagnosis of pathogenic and non-pathogenic Mycobacterium, in suspected BTB cases.

Keywords

References

Aghazadeh, R; Tebianian, M and Mahdavi, M (2016). Evaluation of specific Anti-ESAT-6 antibody in diagnosis of bovine tuberculosis. J. Knowledge Health. 10: 72-78 (in Persian).
Ayele, WY; Neill, SD; Zinsstag, J; Weiss, MG and Pavlik, I (2004). Bovine tuberculosis: an old disease but a new threat to Africa. Int. J. Tuberc. Lung. Dis., 8: 924-937.
Bao, L; Fan, Q; Lu, M and Xia, ZY (2013). Mycobacterium tuberculosis MPT-64 stimulates the activation of murine macrophage modulated by IFN-γ. Eur. Rev. Med. Pharmacol. Sci., 17: 3296-3305.
Baumann, S; Eddine, A and Kaufmann, SH (2006). Progress in tuberculosis vaccine development. Curr. Opin. Immunol., 18: 438-448.
Cho, SN (2007). Current issues on molecular and immunological diagnosis of tuberculosis. Yonsei Med. J., 48: 347-359.
Coler, RN; Skeiky, YA and Ovendale, PJ (2000). Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity. J. Infect Dis., 182: 224-233.
Daniel, TM and Janicki, BW (1978). Mycobacterial antigens: a review of their isolation chemistry and immunological properties. Microbiology. 42: 84-113.
De Kantor, IN and Ritacco, V) 2006(. An update on bovine tuberculosis programmes in Latin American and Caribbean countries. Vet. Microbiol., 112: 111-118.
Dye, C; Garnett, GP; Sleeman, K and Williams, BG (1998). Prospects for worldwide tuberculosis control under the World Health Organization (WHO) DOTS strategy. Lancet. 352: 1886-1891.
Fan, X; Wang, C and Shi, C (2009). An improved whole-blood gamma interferon assay based on the CFP21-MPT64 fusion protein. Clin. Vaccine Immunol., 16: 686-691.
Jolley, ME; Nasir, MS; Surujballi, OP; Romanowska, A; Renteria, TB and De La Mora, A (2007). Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine. Vet. Microbiol., 120: 113-121.
Lange, C and Mori, T (2010). Advances in the diagnosis of tuberculosis. Respirology. 15: 220-240.
Li, H; Ulstrup, JC; Jonassen, TO; Melby, K; Nagai, S and
Harboe, M (1993). Evidence for absence of the mpb-64 gene in some substrains of Mycobacterium bovis BCG. Infect. Immun., 61: 1730-1734.
Melo, E; Souza, I; Ramos, C; Osório, A; Verbisck, N and Araújo, F (2015). Evaluation of the use of recombinant proteins of Mycobacterium bovis as antigens in intradermal tests for diagnosis of bovine tuberculosis. Arch. Med. Vet., 47: 273-280.
Mostafavi-Pour, Z; Hemmati, M; Seghatoleslam, A; Rasti, M; Ebadat, M; Mosavari, N; Habibagahi, M; Taheri, M and Sardarian, A (2011). Expression and purification of recombinant Mycobacterium tuberculosis diagnosis antigens, ESAT-6, CFP-10, and ESAT-6/CFP-10, and their potential use in the diagnosis and detection of tuberculosis. Iran. Red Crescent Med. J., 13: 558-565.
Okada, M and Kobayashi, K (2009). Recent progress in mycobacteriology. Kekkaku. 82: 783-799.
Parkash, O; Singh, B and Pai, M (2009). Regions of differences encoded antigens as targets for immuno-diagnosis of tuberculosis in humans. Scandinavian J. Immunol., 70: 345-357.
Pourakbari, B; Mahmoudi, S; Mamishi, S; Ghazi, M and Hosseinpour Sadeghi, R (2013). Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens. Iran. J. Microbiol., 5: 374-378.
Redchuk, T; Korotkevich, N; Gorbatiuk, O; Gilchuk, P; Kaberniuk, A; Oliynyk, O; Kolibo, D and Komisarenko, S (2012). Expression of Mycobacterium tuberculosis proteins MPT63 and MPT83 as a fusion: purification, refolding and immunological characterization. J. Appl. Biomed., 10: 169-176.
Sambrook, J and Russell, DW (2001). Molecular cloning: a laboratory manual. 3rd Edn., New York: ColdSpringHarbor Laboratory Press. PP: 5-40.
Smith, I (2003). Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol., 16: 463-496.
Souza, II; Melo, ES; Ramos, CA; Farias, TA; Osório, AL; Jorge, KS; Vidal, CE; Silva, AS; Silva, MR; Pellegrin, AO and Araújo, FR (2012). Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis. Springerplus. 1: 77.
Tebianian, M; Moradi, J; Mosavari, N; Ebrahimi, M and Arefpajohi, R (2015). Evaluation of Mycobacterium tuberculosis early secreted antigenic target 6 recombinant protein as a diagnostic marker in skin test. Osong Public Health Res. Perspect., 6: e34-e38.
Van Soolingen, D; De Hass, EWP and Kremer, K (2001). Restriction fragment length polymorphism (RFLP) typing of Mycobacteria. Methods Mol. Med., 54: 165-203.
Wayne, LG and Kubica, GP (1986). The Mycobacteria. In: Sneath, PHA and Holt, JG (Eds.), Bergeys manual of systematic bacteriology. (2nd Edn.), Vol. 2, Baltimore: Williams & Wilkins Co., PP: 1435-1457.
Young, DB (1992). Heat-shock proteins: immunity and autoimmunity. Curr. Opin. Immunol., 4: 396-400.
Zavaran Hoseini, A; Eslami, MB and Jalali, M (1997). Purification antigen isolated from Mycobacterium tuberculosis (H37Rv strain) and their effects on cell-mediated immune responses guinea pigs. M.J.IRI., 10: 291-297.
Zavaran Hoseini, A; Tebianian, M; Ebrahimi, SM; Rezaei Mokaram, A; Taghizadeh, M and Asli, E (2009). Cloning and expression of Mycobacterium tuberculosis ESAT-6 in prokaryotic system. Arch. Razi Inst., 64: 1-7.

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