E2F6基因真核表达质粒的构建与鉴定

Construction and verification of eukaryotic expression plasmid carrying human E2F6 gene

目的构建含E2F6基因的重组pFLAGCMV10质粒并进行鉴定。方法以人全血细胞的总RNA为模板,用RT-PCR方法扩增出E2F6基因。将PCR产物克隆进真核载体pFLAGC-MV10内,构建含E2F6基因的重组真核表达质粒。重组质粒转染293T细胞,用Western blot方法检测E2F6在293T细胞中的表达。结果核酸序列分析的结果表明,克隆的E2F6基因与GenBank中已登记的E2F6基因序列100%同源。Western blot结果显示,在约35-kD位置有目的条带,与预期的重组E2F6蛋白大小一致。结论 成功构建了含E2F6基因的真核表达质粒。

Objective To construct and certificate an eukaryotic expression plasmid carrying human E2F6 gene.Methods E2F6 gene was amplified by RT-PCR with the total RNA of human blood plasma as template.The PCR fragments were cloned into pFLAGCMV10 vector to construct recombinant eukaryotic expression plasmids.293T cells were transfected with the recombinant plasmids.RT-PCR was performed to evaluate the transcription of E2F6 in 293T cells.Western blot was used to detect the expressions of E2F6 in 293T cells.Results The sequence of E2F6 was 100% homology with human E2F6 gene previously registered in GenBank.An interesting band about 35-kD was visible in the result of Western blot,which was consistent with expected size of recombinate protein of E2F6 expressed in 293T cells.Conclusion pFLAGCMV10-E2F6 has been constructed successfully.