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