[Gene-cloning, expression and immunoreactivity detection of Toxoplasma gondii uridine phosphorylase].

OBJECTIVE: To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase (TgUPase), clone, and express TgUPase gene, and analyze its immunoreactivity. METHODS: The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene (GenBank Accession No. DQ385446.1). RT-PCR product was digested with restriction enzyme and ligated into a pET-30a(+) vector. The recombinant plasmid pET-30a(+)-TgUPase was transformed into E. coli DH5alpha and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a(+)-TgUPase was then transformed into E. coli BL21(DE3) and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T. gondii serum was used to confirm the expression of rTgU-Pase and detect its immunoreactivity. RESULTS: Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of M, 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a(+)-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a(+)-TgUPase expressed a soluble protein of His-TgUPase (about Mr 38,000) after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T. gondii serum by Western blotting analysis. CONCLUSION: The recombinant plasmid pET-30a (+)-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.