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Updated: Oct 2, 2026
A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis
Published on: March 24, 2017
[Prokaryotic Expression of Rv0757 Gene in Mycobacterium tuberculosis and the Effect of Its Coding Protein on the
Objective:
To construct recombinant plasmid pET28a-Rv0757 with Mycobacterium tuberculosis (Mtb) Rv0757 gene, and to determine the effect of the protein of Rv0757 gene on ANA-1 cells.
Methods:
We recombined the amplified Rv0757 gene into theprokaryotic plasmid pET28a (+). The expressed product was identified by SDS-PAGE and Western blot. Murine macrophages were treated with purified protein PhoP. Survived cells, lactic dehydrogenase (LDH), nitric oxide (NO), and cell apoptosis were detected after the treatment.
Results:
We successfully constructed the recombinant plasmid pET28a-Rv0757. The target protein was confirmed by SDS-PAGE and Western blot. The protein had no significant effect on cell numbers and LDH activities in the culture supernatant, but it inhibited the release of NO and apoptosis of ANA-1 cells.
Conclusion:
pET28a-Rv0757 plasmid with prokaryotic expression was successfully constructed. The targetprotein has no toxicity on macrophages, but it can inhibit NO release and cell apoptosis of ANA-1.
Insights
The recombinant plasmid pET28a-Rv0757 was successfully constructed. The Rv0757 protein from Mycobacterium tuberculosis inhibits nitric oxide release and apoptosis in ANA-1 cells without toxicity.
Area of Science:
- Molecular Biology
- Immunology
- Microbiology
Background:
- Mycobacterium tuberculosis (Mtb) poses a significant global health challenge.
- Understanding Mtb virulence factors is crucial for developing therapeutic strategies.
- Rv0757 is a gene identified in Mtb with potential roles in host-pathogen interactions.
Purpose of the Study:
- To construct a recombinant plasmid for expressing the Mtb Rv0757 gene.
- To investigate the effects of the Rv0757 protein on ANA-1 macrophage cells.
- To assess the protein's impact on cell viability, nitric oxide production, and apoptosis.
Main Methods:
- Recombinant plasmid pET28a-Rv0757 was constructed by cloning the Rv0757 gene.
- Protein expression was confirmed using SDS-PAGE and Western blot analysis.
- ANA-1 cells were treated with purified Rv0757 protein, and outcomes including cell survival, LDH, NO, and apoptosis were measured.
Main Results:
- Successful construction and verification of the pET28a-Rv0757 recombinant plasmid.
- The expressed Rv0757 protein demonstrated no significant toxicity to ANA-1 cells or impact on cell numbers and LDH activity.
- The Rv0757 protein significantly inhibited nitric oxide release and apoptosis in ANA-1 cells.
Conclusions:
- The prokaryotic expression plasmid pET28a-Rv0757 was successfully generated.
- The Rv0757 protein exhibits a lack of toxicity towards macrophages.
- Rv0757 protein demonstrates a protective effect by inhibiting nitric oxide release and apoptosis in ANA-1 cells.
