弓形虫ROP21基因的原核表达与免疫学鉴定  

Prokaryotic expression and identification of rhoptry protein 21 of Toxoplasma gondii

作　　者：李瑾[1] 孙慧[1] 崔勇[1] 王洪法[1] 刘学峰 于泓 赵桂华[1] 尹昆[1] 仲维霞[1] 徐超[1] 肖婷[1] 魏庆宽[1] 黄炳成 刘功振[1] 

机构地区：[1]山东省医学科学院,山东省寄生虫病防治研究所,山东济宁272033 [2]临沂市畜牧局 [3]平邑县畜牧局

出　　处：《中国病原生物学杂志》2016年第11期1010-1013,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金青年项目(No.31502057)

摘　　要：目的原核表达弓形虫棒状体ROP21基因,并对rROP21蛋白进行免疫学鉴定。方法根据已发表的ROP21基因序列设计引物,通过RT-PCR方法扩增弓形虫RH株的ROP21基因。将ROP21基因克隆到原核表达载体pET-28a(+)后转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导表达目的蛋白,纯化后进行SDS-PAGE分析、Western blot及IFA分析。结果经PCR和酶切鉴定,成功构建重组表达载体。重组质粒转化BL21(DE3)后经IPTG诱导,重组蛋白(rROP21)以包涵体形式高效表达,分子质量单位约为45ku。Western blot显示重组rROP21蛋白能被his标签抗体及弓形虫病患者血清识别。用rROP21免疫小鼠,获得滴度为1∶104的抗体血清。IFA显示弓形虫速殖子体内存在ROP21蛋白。结论构建的重组质粒pET28a-ROP21表达的弓形虫rROP21具有抗性,可作为血清学诊断候选抗原,为进一步研究其生物学功能奠定了基础。To prokaryotically express rhoptry protein ROP21 of Toxoplasma gondii. Methods In accordance with the published sequence of the ROP21 gene,ROP21was amplified with RT-PCR.ROP21 was cloned into the prokaryotic expression vector pET-28a（＋）and then transformed into Escherichia coli BL21（DE3）cells.Expression of the target protein was induced with IPTG,and then the recombinant protein（rROP21）was identified with SDS-PAGE,Western blotting（WB）,and an indirect immunofluorescent assay（IFA）. Results SDS-PAGE indicated that rROP21 was efficiently expressed with a molecular weight of about 45 KD,while Western blotting indicated that rROP21 reacted with histagged antibodies and antibodies from patients with toxoplasmosis.Mice were immunized with the rROP21 protein,resulting in an antibody titer of 1×105.IFA results revealed that antibodies against ROP21 reacted to T.gondii tachyzoites. Conclusion Recombinant rROP21 protein was identified as a potential antigen for serological diagnosis,laying the foundation for further study of its biological function.

关 键 词：弓形虫 棒状体蛋白21 原核表达 Western BLOT 纳虫空泡 

分 类 号：R382.5[医药卫生—医学寄生虫学]