摘要
目的构建基于四环素诱导调控表达系统的刚地弓形虫β-酮脂酰ACP合成酶(FABZ)缺陷株。方法从弓形虫基因组中扩增fabz基因并构建四环素调控诱导表达载体pTetO7-Sag1-FABZ-Ty-DHFR,电穿孔法导入弓形虫TATi株,通过乙胺嘧啶抗性和极限稀释法筛选FABZ缺陷株,蛋白质印迹(Western blotting)检测虫体内附加表位标签Ty的表达。在5×105个FABZ缺陷株弓形虫速殖子培养液中加入脱水四环素(终浓度为1μg/ml),Western blotting检测带有Ty标签的FABZ表达情况。结果筛选获得的缺陷株可表达带有Ty标签的转运肽型FABZ和成熟型FABZ。FABZ缺陷株弓形虫速殖子培养液中加入脱水四环素24 h和48 h后,转运肽型FABZ表达量均较对照组显著降低(P<0.05)。结论构建了由脱水四环素调控表达的弓形虫FABZ缺陷株。
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. 5×10^5 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.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2014年第4期264-267,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学青年基金(No.81301453)
卫生部寄生虫病原与媒介生物学重点实验室开放课题(No.WSBKTKT201302)
江苏大学高级人才启动基金(No.13JDG023
13JDG127)
江苏省研究生创新计划(No.CX10B_282Z)~~
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
Toxoplasma gondii
Fatty acid synthase
Tetracycline inducible expression system
Defective mutant
