Chinese1基因型弓形虫棒状体蛋白ROP16的基因克隆表达及生物信息学分析

发布时间：2018-01-12 09:01

本文关键词：Chinese1基因型弓形虫棒状体蛋白ROP16的基因克隆表达及生物信息学分析　出处：《安徽医药》2015年04期 　论文类型：期刊论文

【摘要】：目的构建弓形虫Chinese 1基因型Tg Ct Wh3(以后均简称Wh3)株棒状体蛋白(rhoptry protein,ROPs)16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至p MD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体p ET-28a和真核表达载体p EGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3D结构。结果各组均PCR扩增出约2.1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体p ET-28a和真核表达载体p EGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以p ET-28a和p EGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。
[Abstract]:Objective to construct the rhoptry protein of Toxoplasma gondii Chinese 1 genotype TG Ct Wh3. The prokaryotic and eukaryotic expression plasmids of ROPs)16 and their 3D structures were designed respectively according to the ROP16 sequence. The gene encoding ROP16 was amplified by PCR from genomic DNA of Toxoplasma gondii Chinese 1 genotype Wh3 strain and cloned into p MD18-T vector. It was identified by PCR and sequencing. The positive clones were subcloned into prokaryotic expression vector p ET-28a and eukaryotic expression vector pEGFP-C2, respectively, and transformed into Escherichia coli BL21 and DH5 伪, respectively. PCR and enzyme digestion were used to identify the inserted sequence of transformed colonies. The recombinant prokaryotic expression strain was induced by IPTG and SDS-PAGE and Western blot were used to analyze the expression of fusion protein. The constructed eukaryotic recombinant plasmid was transfected into 293T cells by liposome and its expression was observed. Results the specific fragment of 2. 1 kb ROP16 gene was amplified by PCR in each group, and the results were correct. Subcloned into prokaryotic expression vector p ET-28a and eukaryotic expression vector p EGFP-C2, respectively. The prokaryotic expression plasmids and eukaryotic expression plasmids of Chinese 1 genotype Toxoplasma gondii corydbody protein ROP16 were successfully constructed. The prokaryotic expression plasmid expressed the fusion protein of ROP16 in Escherichia coli. The eukaryotic expression plasmid was successfully expressed in 293T cells and the 3D structure of ROP16 gene was successfully constructed. Conclusion the vector p ET-28a and p EGFP-C2 are used as vectors. The prokaryotic and eukaryotic recombinant plasmids of ROP16 were constructed and expressed successfully, and the 3D structure of the recombinant plasmids were constructed.
【作者单位】：安徽医科大学基础医学院病原与免疫学实验室;安徽省六安市人民医院检验科;安徽医科大学第一附属医院检验科;安徽医科大学公共卫生学院卫生检验检疫系;安徽医科大学基础医学院寄生虫学教研室;
【基金】：国家重点基础研究发展计划(973计划)(No2010CB530001) 国家青年科学基金(No 81101272)
【分类号】：R382.5;R3416
【正文快照】： (1.安徽医科大学基础医学院病原与免疫学实验室,安徽合肥230032;2.安徽省六安市人民医院检验科,安徽六安237000;3.安徽医科大学第一附属医院检验科,安徽合肥230022;4.安徽医科大学公共卫生学院卫生检验检疫系,安徽合肥230032;5.安徽医科大学基础医学院寄生虫学教研室,安徽合肥

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