摘要
目的构建弓形虫三种不同毒株pMD-18-T/GRA1重组质粒.方法从接种了弓形虫三种不同毒株RH株、桂弓株、B36株的小鼠腹水中收集、纯化虫体,提取基因组DNA.应用PCR技术,分别扩增出三个不同虫株的致密颗粒蛋白1(GRA1)目的基因片段.将此目的基因片段插入PCR产物克隆专用载体pMD-18-T中,转化大肠埃希菌JM109,PCR鉴定其准确性.结果获得弓形虫三种不同毒株GRA1目的基因片段为785 bp.构建了pMD-18-T/GRA1重组质粒,经鉴定与预期理论值相符.结论成功构建了弓形虫三种不同毒株pMD-18-T/GRA1重组质粒.进行弓形虫不同毒株的GRA1基因测序,为研究其同源性及进一步应用研究创造了条件.
Objective To construct a pDM -18 -T/GRA1 recombinant plasmid from three different virulent strains of Toxoplasma gondii. Methods Toxoplasma gondii had been harvested from ascites of mice infected by incubating RH stain, Gui Gong strain and B36 strain intraperitonea, purifying Toxoplasma gondii and preparing genomic DNA. Using PCR, a specific fragment of GRA1 gene was obtained by amplification from three different virulent strains of Toxoplasma gondii. The fragments of PCR products were cloned into a high level sequencing vector pMD -18 -T, and then transferred into JM109, a recombinant pDM - 18 -T/GRA1 was constructed, and it was identified by PCR. Results The size of the amplification GRA1 gene fragment was 785 bp. It was in accord with the expected one. The recombinant pDM - 18 - T/GRA1 was constructed. Conclusion The recombinant sequencing plasmid pDM - 18 - T/GRA1 is successfully constructed. The sequencing of the GRA1 gene from three different virulent strains of Toxoplasma gondii will be further investigated.
出处
《昆明医学院学报》
2006年第4期11-15,共5页
Journal of Kunming Medical College
基金
云南省教育厅科学基金资助项目(03Z500C)
