[Construction and identification of eukaryotic expression vectors of IKKα carrying Ser176/180 phosphorylation site mutants].

OBJECTIVE:To construct eukaryotic expression vectors of the inhibitor of nuclear factor Kappa-B kinaseα (IKKα) containing Ser176/180 phosphorylation site mutants. METHODS:Using PCR, we amplified two IKKα gene fragments, one containing Ser176/180 site mutants and non-sense mutation KpnI and the other with non-sense mutation KpnI. They were subjected to double enzyme digestion, HindIII/KpnI and KpnI/EcoRI, respectively, and then subcloned into pEGFP that had also digested with HindIII/EcoRI beforehand. The location of pEGFP-IKKα (kinase activity, KA)/(kinase death, KD) fusion protein in the eukaryocytes and their effects on P65 nuclear translocation were observed under a confocal microscope. The fusion protein expressions were detected by Western blotting. RESULTS:Sequencing analysis verified the fidelity of all plasmid DNA constructs. PEGFP-IKKα KA was mainly located in nucleus and cytoplasm, and regulated P65 nuclear shuttling. In contrast, pEGFP-IKKα KD was mostly localized in cytoplasm, and did not initiate P65 nuclear translocation. Western blotting confirmed the expressions of the fusion proteins. CONCLUSION:The eukaryotic expression vectors pEGFP-IKKα KA/KD were successfully constructed and well expressed in the eukaryocytes. The fusion proteins were located in different parts of the cells and exerted different effects on P65 nuclear translocation.