刚地弓形虫致密颗粒蛋白GRA7基因的重组、表达及鉴定

发布时间：2018-01-28 02:45

本文关键词： 弓形虫致密颗粒蛋白融合蛋白蛋白纯化　出处：《中国病原生物学杂志》2014年03期 　论文类型：期刊论文

【摘要】：目的构建弓形虫致密颗粒蛋白7(GRA7)基因重组质粒并在大肠埃希菌中进行表达。方法根据GRA7基因序列设计合成引物,用PCR方法从弓形虫基因组DNA中扩增GRA7基因片段,再克隆到pGEX-4T载体中,重组质粒经酶切、PCR鉴定并测序;重组质粒在大肠埃希菌BL21(DE3)中诱导表达,表达产物经SDS-PAGE分析并纯化,Western blot分析其反应原性。结果 GRA7基因PCR产物大小约为714bp,与预期相符;重组质粒经酶切及PCR鉴定构建成功,测序结果与已知序列吻合;重组质粒转化菌经IPTG诱导后表达的GRA7融合蛋白分子质量单位约为53ku,该蛋白可被His标签抗体识别。结论成功重组了弓形虫GRA7基因,表达蛋白具有反应原性,为弓形虫诊断抗原试剂盒的制备奠定了基础。
[Abstract]:Objective to construct the Toxoplasma gondii dense granule protein 7 (GRA7) gene recombinant plasmid and expressed in Escherichia coli. Methods according to the design and synthesis of primers of GRA7 gene sequence, GRA7 gene was amplified from the genomic DNA of Toxoplasma gondii by PCR method, then cloned into pGEX-4T vector. The recombinant plasmid was verified by enzyme digestion, PCR identification and sequencing; the recombinant plasmid in Escherichia coli BL21 (DE3) expression induced by SDS-PAGE, expression analysis and purification, analysis of its reactionogenicity Western blot GRA7. The PCR product was about 714bp in size, in line with expectations; the recombinant plasmid was identified by enzyme digestion and PCR sequencing was constructed successfully, results are consistent with the known sequence of the recombinant plasmid; transformation of bacteria induced by IPTG expression of GRA7 fusion protein molecular weight is about 53ku, the protein was identified by anti His tag antibody. Conclusion recombinant Toxoplasma gondii GRA7 gene expression protein reactionogenicity, It lays the foundation for the preparation of the diagnostic antigen kit for Toxoplasma gondii.

【作者单位】：山东省医学科学院山东省寄生虫病防治研究所;济南大学山东省医学科学院医学与生命科学学院;山东省济宁市第一人民医院;
【基金】：山东省自然科学基金项目(No.2009C03083) 山东省医药卫生科技计划项目(No.2011HW049)
【分类号】：R382
【正文快照】： 弓形虫(Toxoplasma gondii)是一种重要的机会性致病原虫,可寄生在人和许多动物有核细胞中,存活于纳虫泡(parasitophorous vaeuole,pV)的特化空泡内。PV内的致密颗粒细胞器(densegranule organelles)是一种分泌小泡,在修饰调节寄生虫的纳虫泡结构上起着重要作用,并且与虫体在细

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