[Construction, identification and immunoreactivity of Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid]

To construct a Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid and investigate its immunoreactivity.

The ESAT-6 gene fragment amplified from Mycobacterium tuberculosis genome was inserted into pVAX1 vector to construct the recombinant plasmid pVAX1/ESAT-6, which was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Hela cells using Sofast® Transfection reagent, and the cellular expressions of ESAT-6 mRNA and protein were analyzed by RT-PCR and immunofluorescence assay, respectively. The recombinant plasmid pVAX1/ESAT-6 was also transfected into mouse by electronic pulse method, and the mouse serum IFN-γ level and anti-ESAT-6 IgG antibody level were detected by ELISA, mouse lymphocyte proliferation assessed with flow cytometry, and IFN-γ-secreting lymphocytes counted using ELISPOT.

Double restriction-enzyme digestion and sequencing showed that the inserted fragment in the recombinant plasmid pVAX1/ESAT-6 was identical to ESAT-6 gene with an inframe insertion. RT-PCR yielded the target band as expected on agarose gel, and immunofluorescence assay of the transfected cells showed specific green fluorescence signals. The mice transfected with the recombinant plasmid showed significantly elevated serum level of anti-ESAT-6 IgG antibody and increased serum IFN-γ level, spleen cell proliferation, and number of IFN-γ-secreting lymphocytes.

The Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid we constructed can induce high levels of cellular and humoral immunoreactivity in mice.