[Construction of the fusion gene of Hsp65 of Mycobacterium tuberculosis and EGFP and preparation of dendritic cell vaccine against tuberculosis].

AIM

To construct the fusion gene of Hsp65 of Mycobacterium tuberculosis H37Rv and enhanced green fluorescence protein (EGFP) and prepare dendritic cell (DC) vaccine.

METHODS

Hsp65 DNA amplified by PCR was cloned into eukaryotic expression vector EGFP-C1. The recombinant plasmid pEGHsp65 was subsequently transfected into Hela cells, and the transfection rate was determined under confocal laser scanning fluorescence microscope at different times. RT-PCR was used to detect the expression of Hsp65 mRNA in Hela cells. The GM-CSF and IL-4 induced DCs from mouse bone marrow cells were transfected with recombinant plasmid pEGHsp65. Proliferation of unprimed splenocytes activated by transfected DCs was detected by MTT colorimetry.

RESULTS

Restrictive enzyme digestion analysis (EcoR I, Bgl II) confirmed that Hsp65 DNA had been inserted into the vector pEGFP-C1. The recombinant plasmid pEGHsp65 was transfected into Hela cells and the expression of the fusion gene reached peak at the 48 hours after transfection. Expression of Hsp65 mRNA was detected in Hela cells by RT-PCR. DCs transfected with pEGHsp65 could stimulate the proliferation of unprimed splenocytes.

CONCLUSION

The pEGHsp65 fusion gene was successfully constructed and DCs transfected with the pEGHsp65 might be a candidate vaccine for tuberculosis.