Trim5α对TAB2诱导的NFκB启动子荧光素酶报告基因活性的影响

Effects of Trim5α on the activation of luciferase reporter gene containing NFκB promoter induced by TAB2

目的构建人Trim5α的真核表达载体pcDNA3.1(+)-Trim5α,观察其对TAB2诱导的NFκB启动子荧光素酶报告基因活性的影响。方法提取Hela细胞总RNA,用RT-PCR方法扩增出Trim5α基因编码区全长片段,克隆到5`-Flag-pcDNA3.1(+),经酶切、菌落PCR及测序进行鉴定。转染HEK293T细胞,Western blot检测Trim5α蛋白的表达,双萤光素酶报告基因系统检测Trim5α对TAB2诱导的NFκB报告基因活化的影响。结果经鉴定,成功构建Trim5α表达载体,该载体可在HEK293T细胞中表达Trim5α,Trim5α对TAB2诱导的NFκB报告基因活化无显著影响。结论 Trim5α对TAB2诱导的NFκB报告基因活化未见显著影响,初步建立了Trim蛋白对NFκB报告基因影响的功能筛选平台。

We aimed to construct the recombinant eukaryotic expression vector pcDNA3.1（＋）-Trim5α and investigate the effects of Trim5α on activation of luciferase reporter gene containing NFκB promoter induced by adaptor proteins TAB2.The total RNA was isolated from Hela cells.The target sequences were amplified with RT-PCR,and then cloned into 5-Flag-pcDNA3.1（＋） vector.The recombinant vector was confirmed by restriction enzyme digestion,colony PCR and sequencing,and then was transfected into HEK293T cells to detect Trim5α expression by Western blot.Then,the NFκB activities were determined by dual-luciferase reporter assay.The results of restriction enzyme digestion,colony PCR and sequencing confirmed the vector was constructed successfully,and it expressed Trim5α protein in HEK293T cells.However,dual-luciferase assay showed that Trim5α could not effectively blocks TAB2-induced NFκB activation.In conclusion,Trim5α is not involved in negative regulation of TAB2-induced NFκB activation,thus we preliminarily establish a functional screening platform for Trim protein to evaluate its effects on TAB2-induced NFκB activation.