[Cloning and expression of the signaling protein 14-3-3 of Toxoplasma gondii].

OBJECTIVE: To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. METHODS: Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI/Xho I restricted fragments, confirmed by PCR and EcoRI/XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformed into E. coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. RESULTS: The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/Toxo 14-3-3/E. coli BL21 when confirmed by Western blotting. CONCLUSION: The recombinant construction of Toxo 14-3-3 was generated and expression was induced.