真核表达载体pIRES2-AcGFP1-Ascl2的构建及表达

Construction and expression of the eukaryotic expression vector pIRES2-AcGFP1-Ascl2

目的构建真核表达载体pIRES2-AcGFP1-Ascl2,并在真核细胞HEK293T细胞中表达,为今后研究Ascl2基因在Tfh细胞的相关分子机制奠定实验基础。方法 PCR方法扩增健康人外周血单个核细胞中Ascl2基因,连接至pIRES2-AcGFP1质粒的多克隆位点中,构建pIRES2-AcGFP1-Ascl2重组质粒。通过脂质体转染法对HEK293T细胞进行转染。Realtime-PCR及Westernblot方法分析转染pIRES2-AcGFP1-Ascl2重组质粒的HEK293T细胞中Ascl2表达情况。结果经Realtime-PCR及Westernblot方法,证实Ascl2基因能够在HEK293T细胞中正确表达。结论成功建立表达Ascl2基因的pIRES2-AcGFP1-Ascl2重组质粒,并能在HEK293T细胞中表达,为进一步研究Ascl2在自身免疫性疾病中Tfh细胞的相关机制奠定了实验基础。

Objective To construct the pIRES2-AcGFP1-Ascl2 recombinant plasmid which encodes the human Ascl2 gene, and overexpress in human HEK293T ceils. Methods The Ascl2 gene was amplified from the PBMC of the health person by PCR method, then cloned to CDS sites of the pIRES2-AcGFP1 vector.Transfect the pIRES2-AcGFP1-Ascl2 plasmid into HEK293T cell with the lipofectine,then detect the Ascl2 expression in the pIRES2-AcGFP1-Ascl2 transfected HEK293T cell with Realtime-PCR and Westernblot method. Results Realtime-PCR and westernblot demonstrate that pIRES2-AcGFP1-Ascl2 recombinant plasmid had successfully constructed and it can expressed in the human HEK293T cell. Conclusion The recombiant pIRES2-AcGFP1-Ascl2 plasmid had successfully constructed, which is conducive for the follow up research of the function of Ascl2 protein in the Tfh cell in the autoimmune disease.