title: Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.
abstract: OBJECTIVE
  Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies.
  
  
  METHODS
  The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies.
  
  
  RESULTS
  Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein.
  
  
  CONCLUSIONS
  These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
authors.forename: Maryam Mohammadi
authors.initials: MM
authors.lastname: Tashakkori
authors.affiliation: Department of Biotechnology, Razi Vaccine and Serum Research Institute, Karaj, Iran. Electronic address: tebianianm@hotmail.com.
authors.forename: Majid
authors.initials: M
authors.lastname: Tebianian
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Tabatabaei
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
authors.forename: Nader
authors.initials: N
authors.lastname: Mosavari
keywords: Cloning; MPT-64 protein; Mycobacterium bovis; Recombinant protein