[Preparation of an autophagy-targeted antituberculosis DNA vaccine encoding LpqH fused with LC3].

OBJECTIVE

To construct an autophagy-targeted vaccine harboring the genes encoding lipoprotein antigen precursor LpqH from Mycobacterium tuberculosis and microtubule-associated protein light chain-3(LC3), and to investigate its efficacy of inducing and targeting autophagy.

METHODS

The expressions of LC-3 and LC3-LpqH in RAW264.7 cells were detected by Western blotting after transfected with pCMV-LpqH and pCMV-LC3-LpqH plasmids respectively. The pCMV-LC3-LpqH or pCMV-LpqH plasmids were transfected into GFP-LC3-RAW264.7 cells to analyze the localization of LC3-LpqH by immunofluorescence staining.

RESULTS

After transfected with pCMV-LpqH DNA, RAW264.7 cells showed a significant increase of LC-3 amount. The LC3-LpqH fusion protein was also detected in RAW264.7 cells after pCMV-LC3-LpqH transfection and in a dose-dependent manner. Interestingly, LpqH was found to be transported to autophagosomes through the fusion protein, which was demonstrated by the co-localization of GFP-LC3 and LC3-LpqH on autophagosomes.

CONCLUSION

The recombinant plasmid encoding pCMV-LC3-LpqH could enhance the autophagy in vitro, and facilitate the localization of LpqH on autophagosomes. Our study provides a new practical strategy for the development of improved vaccines against Mycobacterium tuberculosis.