Related Experiment Video
Updated: May 27, 2026
Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli
1Gene Expression Laboratory, Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122, India.
Abstract:
Mycobacterium avium subsp. paratuberculosis (Map) contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9 kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9 kDa PPE and 2.2 kDa His-tag), which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1 kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.
Insights
Researchers identified and generated a unique Proline and Glutamic acid rich (PPE) antigen from Mycobacterium avium subsp. paratuberculosis (Map). This recombinant PPE protein is associated with cellular immune responses, offering potential for immunological characterization.
Area of Science:
- Immunology
- Microbiology
- Protein Chemistry
Background:
- Mycobacterium avium subsp. paratuberculosis (Map) possesses PPE antigens, rich in Proline and Glutamic acid, potentially crucial for T cell responses.
- Immunological characterization necessitates the identification and generation of these antigens.
Purpose of the Study:
- To identify and characterize a unique PPE antigen from Map.
- To generate a recombinant PPE protein for immunological studies.
Main Methods:
- Amplification of a unique PPE gene from Map using polymerase chain reaction.
- Cloning the gene into an Escherichia coli expression vector (pQE30 UA) and transformation into E. coli M15.
- Induction of high-level expression of a His-fusion protein, confirmed by immunoblotting and purified using Ni-NTA agarose chromatography.
Main Results:
- A 37.1 kDa His-fusion protein (comprising 34.9 kDa PPE and 2.2 kDa His-tag) was successfully expressed and purified.
- Polyclonal antiserum against the recombinant PPE protein recognized the fusion protein and Map sonicate.
- The recombinant PPE protein induced a significant delayed type hypersensitivity (DTH) skin reaction in Map-sensitized mice.
Conclusions:
- The recombinant PPE protein from Map is linked to cellular immune responses.
- This recombinant protein serves as a valuable tool for the immunological characterization of Map infections.
More Related Videos
06:57Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis
Published on: May 12, 2023
08:51Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling
Published on: June 25, 2015
