关键词: 刚地弓形虫, 脂肪酸代谢酶, 四环素诱导调控表达系统, 缺陷株

Abstract:

  Objective To construct a β-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 limiting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5×105 tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline （ATc, 1 μg/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.

Key words: Toxoplasma gondii, Fatty acid synthase, Tetracycline inducible expression system, Defective mutant