[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.


CONCLUSIONS
The recombinant construction of Toxo 14-3-3 was generated and expression was induced.
Du
Jian
J
Department of Pathobiology, Anhui Medical University, Hefei 230032.
Shen
Ji-long
JL
Wang
Xue-long
XL
Wang
Wei
W
D048948:14-3-3 Proteins; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014122:Toxoplasma / Q000235:genetics*; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis / Q000235:genetics*
D048948:14-3-3 Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014446:Tyrosine 3-Monooxygenase