基于hxgprt^-缺陷的弓形虫顶质体荧光突变株的构建  

Construction of apicoplast fluorescence mutant of hxgprt^- deficient Toxoplasma gondii

作　　者：吴亮[1] 姜旭淦[1] 李礼[1] 鞠爱萍[1] 沈进[1] 傅行礼[1] 陈盛霞[1] 周红[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2012年第1期34-37,42,共5页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省研究生创新计划资助项目(CX10B_282Z);江苏大学科技创新团队项目(2008-018-02)

摘　　要：目的:构建绿色荧光蛋白(green fluorescent protein,GFP)标记顶质体的弓形虫突变株,建立检测顶质体的简便方法。方法:扩增顶质体酰基载体蛋白(acyl carrier protein,ACP)基因并构建转染载体pHX-ACP-GFP,转入弓形虫hxgprt-株速殖子,经霉酚酸和黄嘌呤筛选荧光突变株。激光共聚焦显微镜和蛋白质印迹技术检测突变株ACP-GFP融合蛋白的表达。结果:通过霉酚酸和黄嘌呤筛选,可以在1周内获得突变株。激光共聚焦显微镜下观察到ACP-GFP融合蛋白聚集于顶质体,用抗GFP抗体能检测出ACP的转运肽型和成熟型蛋白。结论:顶质体荧光突变株可清晰标记出顶质体的位置,借助GFP标签即可检测融合蛋白的表达。Objective： To construct Toxoplasma gondii（T.gondii） mutant for expression of green fluorescent protein（GFP） in apicoplast and establish the simple methods for apicoplast detection.Methods： Acyl carrier protein（ACP） gene was amplified from T.gondii genomic DNA and used to construct the transfection plasmid pHX-ACP-GFP,which was transfected into T.gondii tachyzoites.The mutant was selected by mycophenolic acid and xanthine.The expression of GFP was detected by confocal microscope and Western blotting.Results： The mutant was selected by mycophenolic acid and xanthine for one week.The green fluorescent aggregated in apicoplast was observed by confocal microscope.The transit peptide and mature ACPs were detected using anti-GFP tag antibody by Western blotting.Conclusion： Apicoplast could be labeled by ACP-GFP fusion protein which was detected by anti-GFP tag antibody.

关 键 词：弓形虫 顶质体 突变株 绿色荧光蛋白 

分 类 号：R382.5[医药卫生—医学寄生虫学]