Ser176/180磷酸化位点突变的IKKα真核表达载体的构建及鉴定  

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

作　　者：何岩 郑倩倩[1] 朱亚勤[1] 

机构地区：[1]教育部医学细胞生物学重点实验室,细胞病理生物学研究室,中国医科大学基础医学院细胞生物学教研室,辽宁沈阳110001

出　　处：《细胞与分子免疫学杂志》2015年第9期1162-1166,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：辽宁省自然科学基金(20092123);辽宁省教育厅高校科研计划(2009S108)

摘　　要：目的构建Ser176/180磷酸化位点突变的核因子κB抑制剂激酶α(IKKα)真核表达载体。方法通过PCR获得带有IKKαSer176/180突变位点和无义突变KpnⅠ位点的片段,以及带有无义突变KpnⅠ位点的片段,两片段分别经过HindⅢ/KpnⅠ以及KpnⅠ/EcoRⅠ双酶切,载体pEGFP经HindⅢ/EcoRⅠ双酶切后连接。共聚焦显微镜技术观察激活型/死型p EGFPIKKαKA/KD融合蛋白在真核细胞中的定位及其对P65核质易位的影响。Western blot法检测重组蛋白表达。结果测序显示载体构建成功。pEGFP-IKKαKA分布在细胞核和细胞质,并能使P65发生核穿梭。pEGFP-IKKαKD主要分布在细胞质,不能使P65发生核质易位。Western blot结果显示出融合蛋白的特异性条带。结论成功构建IKKα磷酸化位点激活型及死型的真核表达载体,在真核细胞中可有效表达。pEGFP-IKKαKA/KD在真核细胞中蛋白表达呈现不同定位,对下游P65的核质穿梭调控发挥不同效应。Objective To construct eukaryotic expression vectors of the inhibitor of nuclear factor Kappa-B kinase α（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,Hind Ⅲ / Kpn Ⅰ and Kpn Ⅰ / EcoR Ⅰ,respectively,and then subcloned into p EGFP that had also digested with HindⅢ / EcoRⅠ beforehand. The location of p EGFP-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,p EGFP-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 p EGFP-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.

关 键 词：核因子κB抑制剂激酶α 激酶突变型 真核表达载体 

分 类 号：R392.33[医药卫生—免疫学] Q782[医药卫生—基础医学]