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

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-?-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.

SUBMITTER: Mohammadi Tashakkori M

PROVIDER: S-EPMC6056143 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Mohammadi Tashakkori M M Tabatabaei M M Tebianian M M Mosavari N N

Iranian journal of veterinary research 20180101 2

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-stimu ...[more]

PMID: 30046321

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Project description:The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Dataset Information

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

Publications

Production of MPT-64 recombinant protein from virulent strain of <i>Mycobacterium bovis</i>.

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