Subcellular localization of the 54-kDa antigen of Toxoplasma gondii

A 54-kDa protein antigen of Toxoplasma gondii recently was cloned and expressed in Escherichia coli and shown to display immunoprotective properties. To determine the subcellular localization of this antigen, a fusion protein containing the 330 carboxy-terminal residues of the sequence coded by cDNA clone Tg34 was expressed in E. coli, purified, and used to raise antibodies in mice. Western blot analysis confirmed that the resulting antibody reacted with the 54-kDa antigen of T. gondii. Immunofluorescence and immunoelectron-microscopy showed that the antibody reacted with an antigen localized in the rhoptries, 1 of the organelles of the apical complex of the zoites involved in host cell invasion. Western blot studies using a recombinant fusion protein containing the full amino acid sequence encoded by the cDNA clone Tg34 and rhoptry protein-specific monoclonal antibodies (mAbs) previously described showed a reactivity of the recombinant antigen with 2 mAbs (T4 2F8 and T5 2D1) specific for the ROP2 protein, and with a mAb (T3 4A7) directed against an epitope shared by ROP2 and ROP4, but not with a mAb (T2 2H3) specific for the ROP4 protein. We thus conclude that the 54-kDa T. gondii antigen encoded by cDNA clone Tg34 is the previously described rhoptry protein ROP2.