弓形虫棒状体蛋白16的原核表达及多克隆抗体制备  

Prokaryotic expression and polyclonal antibody preparation of rhoptry protein 16 from Toxoplasma gondii

作　　者：刘原[1] 杨华[1] 吴亮[1] 苏丹华[1] 付涛[1] 郭雪[1] 刘曼[1] 姜旭淦[1] 陈盛霞[1] 曹建平[2] 

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]中国疾病预防控制中心寄生虫病预防控制所,上海200025

出　　处：《江苏大学学报（医学版）》2015年第3期251-255,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81301453);卫生部寄生虫病原与媒介生物学重点实验室开放课题(WSBKTKT201302);中国博士后科学基金资助项目(2014M561598);江苏省博士后科研资助计划项目(1402171C);江苏大学高级人才启动基金资助项目(13JDG023;13JDG127)

摘　　要：目的:克隆刚地弓形虫RH株速殖子棒状体蛋白(rhoptry protein,ROP)16基因,并构建ROP16基因的原核表达系统,检测和定位ROP16蛋白的表达。方法:以弓形虫RH株速殖子基因组DNA为模板,PCR扩增弓形虫ROP16基因,克隆至p ET-32a(+)载体,在大肠埃希菌Rosetta中诱导表达。经KCl染色切胶法纯化重组ROP16蛋白,并制备其兔多克隆抗体。采用蛋白质印迹法和间接免疫荧光法检测和定位ROP16在弓形虫速殖子内的表达。结果:成功表达并纯化重组ROP16蛋白,制备了其多克隆抗体。蛋白质印迹法检测出相对分子质量为74 000的特异性条带,间接免疫荧光实验显示ROP16分布于弓形虫速殖子胞质内。结论:经原核表达重组ROP16制备的多克隆抗体能检测和定位ROP16在弓形虫速殖子内的表达。Objective: To clone and construct a recombinant expression plasmid of rhoptry protein( ROP) 16 from tachyzoites of Toxoplasma gondii( T. gondii) RH strain in Escherichia coli,and to determine and locate the expression of ROP 16 in tachyzoites. Methods: The ROP16 gene was amplified from genomic DNA of T. gondii RH strain and cloned into the plasmid p ET-32a( +). The expression of the recombinant p ET32a-ROP16 was induced in E. coli Rosetta strain,and ROP16 was purified by cutting the gel slices with KCl stain. The anti-ROP16 polyclonal antibody was produced in rabbit,and ROP16 in tachyzoites was analyzed by Western blotting and indirect immunofluorescence assay( IFA) respectively. Results:The recombinant plasmid p ET32a-ROP16 was successfully expressed and purified,and the anti-ROP16 polyclonal antibody was obtained. The 74 000 specific band of ROP16 was detected by Western blotting,and the ROP16 was distributed in the cytoplasm of tachyzoites by IFA. Conclusion: The anti-ROP16 polyclonal antibody prepared by recombinant ROP16 can detect and locate the expression of ROP16 in tachyzoites of T. gondii.

关 键 词：刚地弓形虫 棒状体蛋白16 原核表达 KCl染色切胶 

分 类 号：R382.5[医药卫生—医学寄生虫学] R392[医药卫生—基础医学]