大鼠源肺孢子菌p55基因片段真核表达质粒的构建及表达

目的构建大鼠源肺孢子菌抗原p55基因片段真核表达质粒,并研究该质粒在真核细胞COS-7中的有效表达。方法以含有p55全长基因的质粒为模板,通过PCR扩增p55基因片段,克隆至pGEM-T载体中,经酶切后再将p55基因重组至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)-582,经限制性内切酶酶切分析及测序鉴定正确后,应用脂质体转染技术转染入COS-7细胞,RT-PCR检测其基因转录情况,SDS-PAGE鉴定其表达相应的目的蛋白质情况。结果成功扩增了p55基因片段,酶切和测序证明正确构建了重组真核表达质粒pcDNA3.1(+)-582,RT-PCR和SDS-PAGE方法证实该片段能在COS-7细胞中有效表达目的蛋白质。结论成功构建了大鼠源肺孢子菌p55基因片段的真核表达质粒载体,并在COS-7细胞中表达,为进一步进行核酸疫苗的研究奠定了基础。

Construction and expression of the eukaryotic expression vector containing the p55 gene fragment of rat Pneumocystis

To construct the eukaryotic expression plasmid containing the p55 gene fragment of Pneumocystis and to investigate the efficient expression in COS-7 cells,the gene fragment conaining the whole length of p55 gene was used as template to amplify this fragment with PCR and the amplified fragment was then cloned to vector pGEM-T.After enzyme digestion,p55 gene was cloned to the eukaryotic expression vector pcDNA3.1(+)to construct the plasmid pcDNA3.1(+)-582.This plasmid was then transfected to the eukaryotic ex...