Heterologous Expression of a Gene Encoding a 35 kDa Protein of Mycobacterium Avium Paratuberculosis in Escherichia Coli

Vet Res Commun. 2004 Apr;28(3):209-24. doi: 10.1023/b:verc.0000017371.68083.04.

Abstract

The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, Bacterial / biosynthesis*
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / immunology
  • Antigens, Bacterial / isolation & purification
  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology
  • Bacterial Proteins / isolation & purification
  • Blotting, Western
  • Cloning, Molecular
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Guinea Pigs
  • Hypersensitivity, Delayed / immunology
  • Mycobacterium avium subsp. paratuberculosis / genetics*
  • Mycobacterium avium subsp. paratuberculosis / immunology
  • Mycobacterium avium subsp. paratuberculosis / metabolism
  • Polymerase Chain Reaction
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Subcellular Fractions / microbiology
  • Surface Properties

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • DNA, Bacterial
  • Recombinant Proteins