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Updated: May 27, 2026
Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria
Published on: December 8, 2023
[Establishment of RAW264.7 cell line stably expressing Mycobacterium tuberculosis protein ESAT-6]
Hao Li1, Ying Yin, Dayong Dong
1State Key Laboratory ofPathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China.
Abstract:
For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.
Insights
Researchers developed a stable macrophage cell line expressing the Mycobacterium tuberculosis ESAT-6 protein. This tool aids future studies on how ESAT-6 influences macrophage function and tuberculosis pathogenesis.
Area of Science:
- Immunology
- Microbiology
Context:
- Macrophages play a crucial role in the immune response to Mycobacterium tuberculosis (Mtb).
- The Mtb secretory protein ESAT-6 is a key virulence factor influencing host-pathogen interactions.
- Understanding ESAT-6's effects on macrophage function is vital for developing effective tuberculosis therapies.
Purpose:
- To establish a stable RAW264.7 macrophage cell line expressing the enhanced green fluorescent protein (EGFP)-ESAT-6 fusion protein.
- To investigate the gene and protein expression of EGFP-ESAT-6 in the engineered cell line.
- To provide a valuable tool for future research into the mechanisms of ESAT-6 in modulating macrophage functions.
Summary:
- RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 or pEGFP-C1 using liposomes.
- Stable cell lines expressing EGFP-ESAT-6 or EGFP were selected using G418.
- RT-PCR, fluorescence microscopy, and Western blotting confirmed stable integration and expression of the EGFP-ESAT6 fusion gene and protein.
- The study successfully created a tool for future investigations into ESAT-6's role in macrophage modulation.
Impact:
- Provides a robust cellular model for studying the impact of ESAT-6 on macrophage biology.
- Facilitates research into the molecular mechanisms by which Mtb evades host immunity.
- Contributes to the ongoing effort to understand tuberculosis pathogenesis and identify new therapeutic targets.
