【摘要】 目的构建弓形虫致密颗粒蛋白GRA2的真核表达重组质粒。方法设计合成GRA2引物,运用PCR方法扩增其基因片段,经克隆至pMD18-T载体后,亚克隆至真核表达质粒pcDNA3.1(-)而构建重组表达质粒pcDNA3.1-GRA2。脂质体法将构建的重组质粒转染HFF细胞,RT-PCR法检测转染细胞中GRA2的表达情况。结果PCR扩增GRA2基因序列正确,构建的重组表达质粒pcDNA3.1-GRA2经PCR、EcoRⅠ/HindⅢ双酶切和测序鉴定正确;转染GRA2基因的细胞,RT-PCR可见目的条带。结论成功获得真核表达重组质粒pcDNA3.1-GRA2,为进一步研究弓形虫疫苗的免疫保护性奠定基础。更多还原

【Abstract】 Objective To construct an eukaryotic expression recombinant plasmid containing dense granule antigen 2(GRA2) of Toxoplasma gondii. Methods GRA2 gene fragment was amplified by PCR with primers and cloned into pMD18-simple-T vector, then inserted into pcDNA3.1(-) to generate eukaryotic expression plasmid pcDNA3.1-GRA2. The recombinant plasmid was transfected into HFF cells by LipofectamineTM 2000. And expression of GRA2 in cells was detected by RT-PCR. Results The gene fragment encoding GRA2 was amplified correctly by PCR. Enzyme digestion, PCR analysis and identical sequencing showed that the recombinant eukaryotic expression plasmid pcDNA3.1-GRA2 was right constructed. RT-PCR analysis of cells transfected GRA2 gene displayed positive bands. Conclusion Recombinant eukaryotic expression plasmid of GRA2 has been successfully obtained, suggesting a foundation for the further immunity studies on the vaccine development for T. gondii infections.更多还原