刚地弓形虫RH株ROP11基因的克隆、真核表达载体的构建及表达.docVIP

刚地弓形虫RH株ROP11基因的克隆、真核表达载体的构建及表达 张晓磊,张进顺*,朱晓波,王春苗,贾晓晖,贾天军,徐云鹏,高雪 【摘要】 目的 构建刚地弓形虫（Toxoplasma gondii）棒状体蛋白11的真核表达重组质粒并使其在真核细胞中表达目的蛋白。方法 设计合成弓形虫ROP11引物，运用RT-PCR方法扩增，其基因片段克隆至真核表达质粒pcDNA3.1（+），从而构建重组表达质粒pcDNA3.1-ROP11。将重组质粒转染HeLa 细胞，间接免疫荧光法检测细胞内蛋白表达情况。结果 RT-PCR扩增弓形虫ROP11基因序列正确，构建的重组质粒pcDNA3.1-ROP11经PCR、EcoRI和NotI双酶切和测序鉴定正确。重组质粒转染的HeLa 细胞胞浆观察到黄绿色荧光，对照组无荧光。结论 成功获得了真核表达质粒pcDNA3.1-ROP11，且能够在真核细胞中表达目的蛋白,为进一步研制弓形虫疫苗的免疫保护性奠定基础。 【关键词】 刚地弓形虫；ROP11; 真核表达质粒 河北省高等学校科学技术研究重点项目（No. ZH2012010） 作者单位：河北北方学院病原生物与免疫学研究所，张家口 075000 * 通信作者，E-mail：wdzjs@ Gene-cloning，Eukaryotic expression vector-constructing and expressing the Rhoptry Protein 11 of RH strain of Toxoplasma gondii Zhang Xiaolei, Zhang Jinshun*,Zhu Xiaobo,Wang Chunmiao, Jia Xiaohui, Jia Tianjun,Xu Yunpeng,GaoXue 【Abstract 】 Objective To construct an eukaryotic expression recombinant plasmid containing rhoptry protein 11(ROP11)of Toxoplasma gondii and make recombinant plasmid can express ROP11 protein in eukaryotic system. Methods ROP11 gene fragment was amplified by RT-PCR with primers and cloned into pcDNA3.1（+） to construct eukaryotic expression plasmids pcDNA3.1-ROP11. then transfected into HeLa cells． The expressed protein was identified by indirect immunofluorescence Assay．Results The gene fragment encoding ROP11 was amplified correctly by RT-PCR. The recombinant eukaryotic expression plasmid pcDNA3.1-ROP11 was accredited and confirmed to be right by colony-PCR, double restriction enzyme digestion, Sequencing. ROP11 protein expressed in the HeLa cells. Conclusions The successful construction of recombinant eukaryotic expression plasmid of ROP11 and expression ROP11 protein in eukaryotic system has laid foundation for further development of immune protective vaccine against Toxoplasma gondii infections. 【Key words】 Toxoplasma gondii；Rhoptry protein 11；Eukaryotic expression plasmid Supported by Science And Technology Research Key Project of Hebei Province Higher School(No. ZH2012010) * Corresponding author, E-mail：wdzjs@ 刚地弓形