弓形虫速殖子特异表达蛋白SAG1启动子的克隆及鉴定

Cloning and Identification of Promoter of Special Expression Protein SAG1 of Toxoplasma gondii Tachyzoite

目的克隆弓形虫速殖子特异表达蛋白SAG1的启动子序列,为弓形虫基因操作提供工具。方法应用PCR方法扩增弓形虫速殖子特异表达蛋白SAG1的5′非编码区、致密颗粒蛋白GRA2的3′编码区和红色荧光蛋白(RFP)基因,并构建重组质粒pMD/SAG-RFP-GRA,经电穿孔法将其转染弓形虫速殖子,倒置荧光显微镜观察RFP的表达。结果重组质粒pMD/SAG-RFP-GRA经双酶切和测序证明构建正确,质粒转染弓形虫速殖子24h,荧光显微镜下可观察到红色荧光,表明克隆的SAG1启动子具有转录活性。结论已成功克隆了弓形虫速殖子特异表达蛋白SAG1的启动子序列,并能介导外源基因在弓形虫体内的表达。

Objective To clone the promoter sequence of special expression protein SAG1 of Toxoplasma gondii tachyzoite and provide a tool for genetic manipulation of Toxoplasma gondii.Methods The non-encoding region at 5＇-terminus of SAG1,the encoding region of 3＇-terminus of dense grandule protein 2（GRA2）and red fluorescent protein（RFP）gene were amplified by PCR for construction of recombinant plasmid pMD/SAG-RFP-GRA which was transfected to Toxoplasma gondii tachyzoite by electroporation.The expression of RPF was observed under inverted fluorescent microscope.Results Both restriction analysis and sequencing proved that recombinant plasmid pMD /SAG-RFP-GRA was constructed correctly.Red fluorescence was observed in the Toxoplasma gondii tachyzoite 24 h after transfection with the recombinant plasmid,indicating transcription activity of the cloned promoter of SAG1.Conclusion The promoter sequence of SAG1 was successfully cloned,which could mediate the expression of exogenous gene in Toxoplasma gondii tachyzoite.