[Construction of beta-hydroxyacyl-acyl carrier protein dehydratase mutant of Toxoplasma gondii by tetracycline inducible expression system].

OBJECTIVE

To construct a beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ) mutant of Toxoplasma gondii with tetracycline inducible expression system.

METHODS

The fabz gene was amplified from T. gondii genomic DNA, and then used to construct the tetracycline inducible expression vector pTetO7-Sag1-FABZ-Ty-DHFR. The vector was transfected into TATi strain by electroporation. The FABZ defective mutant was selected by pyrimethamine and limitting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5 x 10(5) tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline (ATc, 1 microg/ml) for 24 h and 48 h, respectively. The expression of Ty-tagged FABZ protein in the mutant was detected by Western blotting.

RESULTS

The mutant could express the transit peptide (t-FABZ) and mature FABZ (m-FABZ) with the Ty-epitope tag. After ATc added in culture medium for 24 h and 48 h, the expression of t-FABZ in the mutant decreased significantly (P<0.05).

CONCLUSION

The FABZ mutant is constructed with a tetracycline inducible expression system.