Identification and molecular cloning of the Neospora caninum SAG4 gene specifically expressed at bradyzoite stage

Here, we identify and clone the Nc SAG4 gene, orthologue to the Toxoplasma gondii Tg SAG4 gene, and the first reported gene to be expressed specifically during the Neospora caninum bradyzoite stage. To isolate Nc SAG4, we designed degenerate oligonucleotides based on the TgSAG4 protein amino acid sequence. A 312-bp DNA fragment was amplified by PCR from N. caninum genomic DNA, whose sequence showed 65% identity to Tg SAG4 gene over 257 bp. Nc SAG4 gene sequence was obtained by PCR genome walking. Nucleotide sequencing of amplified DNA fragments showed a single uninterrupted 522-bp ORF that encoded a 173-amino-acid protein with a predicted molecular mass of 18,394 Da, with 69% similarity to the TgSAG4 antigen. A 28-residue putative signal peptide was found at the NH 2-terminus, followed by a strongly hydrophilic region. An amino acid motif for a phosphatidylinositol glycan anchor was identified at the COOH-terminus. The NcSAG4 protein lacking the putative signal peptide at the NH 2-terminus was expressed in Escherichia coli and was recognized in western blot by sera from congenitally infected cattle. A mouse polyclonal anti-rNcSAG4 serum was produced for immunofluorescence studies, and revealed stage-specific NcSAG4 antigen expression in in vitro-cultured bradyzoites. Real-time reverse transcription-PCR analysis with samples from in vitro stage-conversion assay showed increasing levels of Nc SAG4 transcript over time, suggesting a developmental upregulation of this gene.