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. 2018 Spring;19(2):108-112.

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

M Mohammadi Tashakkori  1 , M Tabatabaei  2 , M Tebianian  3 , N Mosavari  3
Affiliations

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

M Mohammadi Tashakkori et al. Iran J Vet Res. 2018 Spring.

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: Cloning; MPT-64 protein; Mycobacterium bovis; Recombinant protein.

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Figures

Fig. 1
Fig. 1
Amplifications of mpt-64 gene from M. bovis genome by PCR. Lane 1: DNA ladder 100 bp (Fermentas, #SM0248), Lane 2: PCR product and Lane 3: Negative control
Fig. 2
Fig. 2
Restriction analysis of recombinant plasmid pQE30-mpt64 by HindIII and BamHI. Two separated fragments were seen. Lower is ~680 bps band of the mpt-64 gene together with a part of the pQE30 backbone and upper is ~3.4 kbp band for the remaining backbone of pQE30 vector in lane 2. Lane 1: Ladder fermentas, Lane 2: Digested with BamHI/HindIII, Lane 3: Single digested, and Lane 4: Undigested
Fig. 3
Fig. 3
Expression of the MPT-64 recombinant protein. Distinct band around 24 kDa was observed in Lane 5, after 3 h induction with IPTG 1 mM. Lane 1: Before induction with IPTG (T0), Lane 2: After 1 h induction with IPTG (T1), Lane 3: Supernatant, after 3 h induction with IPTG, Lane 4: After 2 h induction with IPTG (T2), Lane 5: After 3 h induction with IPTG (T3), and Lane 6: Protein ladder (Fermentas, #SM0661
Fig. 4
Fig. 4
Purification of the protein MPT-64 with Ni-NTA. Lanes 1, 2, 3: Before purification, Lane 4: Washing with B buffer, Lane 5: Washing with C1 buffer, Lane 6: Washing with C2 and C3 buffers, Lane 7: Washing with C4 and C5 buffers, Lane 8: Washing with wash buffer, Lane 9: After purification with Elution buffer, and Lane 10: After purification with Elution and EDTA buffer
Fig. 5
Fig. 5
Western blotting to confirm the recombinant protein MPT-64 with different concentrations of purified protein. Lane 1: Pure protein, Lane 2: 1/2 and lane 3: 1/4, and Land 4: Protein ladder
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