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Expression and purification of a gene encoding a 9.7 kDa PE protein of Mycobacterium avium subsp. paratuberculosis
S Chandra Sekar1, P P Goswami2, R Deb1
1Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, 243122, Uttar Pradesh, India.
Abstract:
Mycobacterium avium subsp. paratuberculosis (Map) contains PE family antigens which are Proline and glutamic acid rich and may play important role as T-cell antigens. In the present study, the Map 1507 ORF encoding 9.7 kDa PE protein was amplified by polymerase chain reaction and cloned into E. coli vector pQE30 UA. The recombinant plasmid designated as pQ PE was transformed into E. coli M15 cells and induced with IPTG revealed the high level expression of 11.9 kDa His-fusion protein as estimated by migration in 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant PE protein was purified by Ni-NTA agarose chromatography. Polyclonal antibodies raised against purified recombinant PE protein reacted with expressed PE protein as well as with Map sonicate. The recombinant PE protein was also recognized by serum from goat with clinical paratuberculosis. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PE protein of Map was associated with T-cell response.
Insights
Mycobacterium avium subsp. paratuberculosis (Map) PE antigens are crucial for T-cell responses. This study shows recombinant PE protein elicits a significant T-cell mediated immune response in paratuberculosis models.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease in ruminants.
- PE family antigens are proline and glutamic acid-rich proteins implicated in T-cell responses.
Observation:
- The Map 1507 ORF encoding a PE protein was amplified and cloned into an E. coli expression vector.
- High-level expression of an 11.9 kDa His-fusion protein was achieved and purified using Ni-NTA chromatography.
- Polyclonal antibodies recognized the recombinant PE protein and Map sonicate.
Findings:
- The recombinant PE protein was recognized by serum from goats with clinical paratuberculosis.
- The purified PE protein elicited significant delayed-type hypersensitivity (DTH) skin reactions in Map-sensitized mice.
- These results indicate the recombinant PE protein is associated with T-cell mediated immunity.
Implications:
- The recombinant PE protein shows potential as a diagnostic marker for paratuberculosis.
- This PE antigen could be a target for developing subunit vaccines against Map infections.
- Further research into Map PE antigens may reveal new insights into host-pathogen interactions.
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