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

作　　者：安娜[1] 张瑞岩[2] 赵志远[3] 商立民[2] 刘全[2] 魏峰[1] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]解放军军事医学科学院军事兽医研究所,长春130062 [3]长春祈健生物制品有限公司市场营销部,长春130012

出　　处：《中国生物制品学杂志》2010年第6期602-604,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助(30972178)

摘　　要：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.

关 键 词：弓形虫病 速殖子 启动区(遗传学) 克隆 

分 类 号：R382.5[医药卫生—医学寄生虫学] Q789[医药卫生—基础医学]