MOLECULAR CHARACTERIZATION OF A 65-KILODALTON TOXOPLASMA-GONDII ANTIGEN EXPRESSED ABUNDANTLY IN THE MATRIX OF TISSUE CYSTS

We describe the cloning and characterization of a novel antigen expressed in the bradyzoite stage of Toxoplasma gondii. A cDNA library was constructed in bacteriophage lambda gt11 Sfi-Not using messenger RNA molecules isolated from cysts of the ME49 strain of T. gondii. The recombinant phage library was subjected to screening with polyclonal antibodies against bradyzoite antigens. This screening identified a recombinant antigen that was recognized strongly by polyclonal antibodies against bradyzoite antigens as well as by sera from mice chronically infected with T. gondii. The native antigen is a protein of 65 kDa that localized to the matrix of the cyst and the cyst wall surrounding the bradyzoites. The antigen was found to be expressed abundantly in cysts but could not be detected in tachyzoites or within the parasitophorous vacuole of tachyzoite infected host cells. Genomic and cDNA sequence of the gene revealed an open reading frame encoding 452 amino acids interrupted by 2 introns: a 503-bp intron located in the 5' untranslated region preceding the protein coding sequence and a 110-bp intron located 95 bp downstream of the first ATG.