Cloning and Expression of the Signaling Protein 14-3-3 of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

ABSTRACT

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 transformd 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.