Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii

Abstract

Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+RNA was made in lambda gt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-IN, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was in great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.