Journal of Southern Medical University ›› 2013, Vol. 33 ›› Issue (07): 945-.
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Abstract: Objective To construct a Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid and investigate itsimmunoreactivity. Methods The ESAT-6 gene fragment amplified from Mycobacterium tuberculosis genome was inserted intopVAX1 vector to construct the recombinant plasmid pVAX1/ESAT-6, which was identified by restriction enzyme digestion andsequencing. The recombinant plasmid was transformed into Hela cells using Sofast® Transfection reagent, and the cellularexpressions of ESAT-6 mRNA and protein were analyzed by RT-PCR and immunofluorescence assay, respectively. Therecombinant plasmid pVAX1/ESAT-6 was also transfected into mouse by electronic pulse method, and the mouse serum IFN-γlevel and anti-ESAT-6 IgG antibody level were detected by ELISA, mouse lymphocyte proliferation assessed with flowcytometry, and IFN-γ-secreting lymphocytes counted using ELISPOT. Results Double restriction-enzyme digestion andsequencing showed that the inserted fragment in the recombinant plasmid pVAX1/ESAT-6 was identical to ESAT-6 gene withan inframe insertion. RT-PCR yielded the target band as expected on agarose gel, and immunofluorescence assay of thetransfected cells showed specific green fluorescence signals. The mice transfected with the recombinant plasmid showedsignificantly elevated serum level of anti-ESAT-6 IgG antibody and increased serum IFN-γ level, spleen cell proliferation, andnumber of IFN-γ-secreting lymphocytes. Conclusion The Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid weconstructed can induce high levels of cellular and humoral immunoreactivity in mice.
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https://www.j-smu.com/EN/Y2013/V33/I07/945