Construction, expression and preliminary immunological evaluation of a DNA plasmid encoding the GRA2 protein of toxoplasma gondii

ABSTRACT

Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious medical problems in immunocompromised individuals and fetus. The infection also causes considerable economic loss because of abortion in livestock, mostly in sheep and goats. DNA vaccination may be a powerful approach against intracellular parasites such as Toxoplasma gondii. The goal of this study was to construct and evaluate the functionality of an eukaryotic expression plasmid pRC/CMV-GRA2, harboring dense granule antigen-2 [GRA2] gene of T. gondii and to perform preliminary studies on its immunogenicity in a mouse model. The GRA2 complete cDNA was inserted in PCR2.1 plasmid, sequenced, then cut and inserted in pRC/CMV plasmid, to produce the recombinant plasmid pRC/CMV-GRA2 [pGRA2]. To verify that the plasmid construct pGRA2 was capable of expressing GRA2 in mammalian cells, it was transfected into 293-T cells, an embryonic kidney cell line. Western-blot analysis of the transfected cells using a monoclonal antibody specific for GRA2 indicated specific expression of GRA2 protein. CBA/J mice were subcutaneously immunized three times with 100 micro g of pGRA2 plasmid. The obtained sera recognized GRA2 that is shown by Western-blotting. These findings indicate that pGRA2 plasmid directs high-level expression of antigenic GRA2 protein in mammalian cells and is immunogenic in CBA/J mice