弓形虫表面抗原1、棒状体蛋白18真核表达载体的构建与体外表达  

Construction and in vitro expression of Toxoplasma gondii SAG1,ROP18 eukaryotic expression plasmids

作　　者：吴腊梅[1] 吴婷[1] 吴亮[1] 王晓[1] 苏丹华[1] 刘原[1] 陶思佳[1] 魏逸青 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246-250,255,共6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:构建刚地弓形虫(Toxoplasma gondii)表面抗原1(surface antigen 1,SAG1)及棒状体蛋白18(rhoptry protein 18,ROP18)的真核表达重组质粒,并在真核细胞中表达目的蛋白。方法:设计SAG1、ROP18的特异引物,采用RCR技术从弓形虫RH株基因组DNA中扩增编码SAG1、ROP18基因片段,经克隆至p TG19-T载体后,亚克隆至真核表达载体p3×FLAG-Myc-CMVTM-24,构建真核表达重组质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-MycCMVTM-24-ROP18。将构建好的真核表达重组质粒转染人肾上皮细胞系293-T细胞,分析转染细胞的表达情况。结果:PCR扩增弓形虫SAG1和ROP18基因片段分别为1 011 bp和1 665 bp,与预期大小相符。重组质粒经PCR、酶切和测序鉴定结果均正确,通过RT-PCR和蛋白质印迹技术鉴定出重组质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-Myc-CMVTM-24-ROP18在293-T细胞中表达。结论:成功构建了真核表达质粒p3×FLAG-Myc-CMVTM-24-SAG1和p3×FLAG-Myc-CMVTM-24-ROP18,该重组质粒能够在真核细胞中表达目的蛋白。Objective: To construct two recombinant eukaryotic expression plasmids containing the surface antigen( SAG) 1 and rhoptry protein( ROP) 18 gene of Toxoplasma gondii,and express SAG1 and ROP18 proteins in the eukaryotic system. Methods: The gene fragments encoding SAG1 and ROP18 were amplified by PCR with special primers from Toxoplasma gondii genomic DNA respectively,and were cloned into p TG19-T vector. The right gene fragments were subcloned into p3 × FLAG-Myc-CMVTM-24 vector to construct the eukaryotic expression plasmids p3 × FLAG-Myc-CMVTM-24-SAG1 and p3 × FLAG-Myc-CMVTM-24-ROP18. The recombinant plasmids were transfected into human renal epithelial 293-T cell lines respectively,and the expression of SAG1 and ROP18 in transfected cells were detected by RT-PCR and Western blotting. Results: The gene fragments encoding SAG1,ROP18 were 1 011 bp and 1 665 bp respectively,which were consistant with the expected size. The analysis of enzyme digestion,PCR and sequencing showed that the recombinant eukaryotic expression plasmids p3 × FLAG-Myc-CMVTM-24-SAG1,p3 × FLAG-Myc-CMVTM-24-ROP18 were constructed correctly. RT-PCR and Western blotting analysis of cells transfected SAG1,ROP18 gene displayedpositive bands. Conclusion: Recombinant eukaryotic expression plasmids of SAG1,ROP18 have been successfully constructed,the recombinant plasmids can express SAG1,ROP18 proteins in a eukaryotic system.

关 键 词：刚地弓形虫 SAG1 ROP18 真核表达质粒 

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