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Updated: Oct 5, 2025
Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis
Published on: September 26, 2025
[Prokaryotic expression and identification of PPE15 protein from Mycobacterium tuberculosis and preparation of rabbit
Tao Xu1, Minying Li2, Chutong Wang3
1Department of Clinical Laboratory, School of Laboratory Medicine, Department of Immunology, Anhui Province Key Laboratory of Immunology in Chronic Diseases, Bengbu 233030, China.
Abstract:
Objective To clone, express and purify PPE15 recombinant protein from Mycobacterium tuberculosis (H37Rv), as well as prepare and characterize its rabbit polyclonal antibody. Methods By The PPE15 gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, and the His-tagged prokaryotic PPE15 prokaryotic expression plasmid pET28a-PPE15 was constructed by homologous recombination cloning technique, and transformed into E. coli BL21 (DE3). PPE15 expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Recombinant PPE15 was identified by SDS-PAGE, and further purified by affinity chromatography with a Ni-NTA column. The renaturation purified PPE15 protein was used to immunize New-Zealand rabbit to prepare polyclonal antibodies. The antibody specificity was analyzed by Western blot analysis, and antibody titer was determined by indirect ELISA. Results Recombinant prokaryotic PPE15 protein was successfully expressed and purified with a molecular weight of 38 kDa. The purified PPE15 protein exhibited positive reaction with the serum of TB patients and the PPE15 protein, the titer of the polyclonal antibodies reaches more than 1:1 300 480. Conclusion The recombinant protein PPE15 was successfully expressed and purified, and high titer rabbit-derived polyclonal antibody was prepared which provided an experimental basis for further functional studies of PPE15 protein.
Insights
Researchers successfully produced and purified the PPE15 recombinant protein from Mycobacterium tuberculosis. They also developed a high-titer rabbit polyclonal antibody, crucial for future functional studies of PPE15 protein in tuberculosis research.
Area of Science:
- Microbiology
- Immunology
- Protein Expression
Background:
- Tuberculosis (TB) remains a significant global health challenge.
- Understanding Mycobacterium tuberculosis (Mtb) proteins like PPE15 is vital for developing new diagnostics and therapeutics.
- Recombinant protein expression and antibody generation are key tools in molecular biology research.
Purpose of the Study:
- To clone, express, and purify the PPE15 recombinant protein from Mycobacterium tuberculosis (H37Rv).
- To prepare and characterize rabbit polyclonal antibodies against the PPE15 protein.
- To establish an experimental basis for further functional studies of PPE15.
Main Methods:
- PPE15 gene amplification from Mtb H37Rv genome using PCR.
- Construction of a His-tagged prokaryotic expression plasmid (pET28a-PPE15) via homologous recombination.
- Expression in E. coli BL21 (DE3) induced by IPTG, followed by purification using Ni-NTA affinity chromatography.
- Antibody production in New Zealand rabbits, with specificity confirmed by Western blot and titer determined by indirect ELISA.
Main Results:
- Successful expression and purification of recombinant PPE15 protein with an approximate molecular weight of 38 kDa.
- The purified PPE15 protein showed positive reactivity with serum from TB patients.
- High-titer rabbit polyclonal antibodies were generated, with titers exceeding 1:1,300,480.
Conclusions:
- Recombinant PPE15 protein from Mycobacterium tuberculosis was successfully expressed and purified.
- A high-titer rabbit polyclonal antibody against PPE15 was successfully prepared.
- This work provides a foundation for in-depth functional investigations of the PPE15 protein.
