IKKβ真核表达载体的构建及表达

Construction of eukaryotic vector of inhibitor of kappa B kinaseβ and expression in HEK293 cells

目的构建核转录因子κB抑制蛋白激酶(inhibitor of kappa B kinase,IKK)β真核表达载体,以便进一步探讨其生物学作用。方法从人正常肝细胞L02中提取RNA,并利用IKKβ特异性引物通过RT-PCR方法扩增了IKKβcD-NA,并克隆入pcDNA3载体中,构建重组载体pcDNA3-IKKβ。将重组载体转染入HEK293细胞中,并通过免疫印迹方法检测IKKβ的表达。结果酶切及测序结果显示,重组质粒构建成功;免疫印迹结果显示,瞬时转染重组质粒的人胚肾细胞HEK293中可有效表达IKKβ。结论本研究成功构建了IKKβ真核表达载体,并能够在真核细胞中进行表达,为进一步研究IKKβ的生物学功能奠定了基础。

Objective Construct the inhibitor of kappa B kinase β recombinant eukaryotic expression vector,in order to further investigate the biological function of IKKβ.Methods Extract total RNA from human liver cell L02 and amplify IKKβ cDNA by RT-PCR using specific primers.Then the cDNA was inserted into vector pcDNA3 to construct the recombinant vector pcDNA-IKKβ,then the recombinant vector was transfected into human embryonic kidney cell HEK293 and the protein expression was detected by western blot.Results Enzyme restriction and sequencing data indicated that the recombinant vector was constructed exactly,western blot result shown that the IKKβ recombinant eukaryotic expression vector can efficiently express IKKβ in HEK293 cells.Conclusion The eukaryotic expression vector pcDNA3-IKKβ is constructed and expresses in HEK293 cells successfully,which establish the foundation for future research on IKKβ gene function.