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目的:构建热休克转录因子4b(Hsf4b)的真核表达载体,探讨MAP激酶P38对其磷酸化调控作用。方法:用人心脏cDNA文库为模板,应用囊括Hsf4b全长的引物进行PCR并在Hsf4b cDNA的N-端加入Flag标签。将PCR产物经KpnI和EcoRI酶切后,与该二酶线性化pcDNA3.0质粒一起连接获得重组质粒pcDNA-Flag-Hsf4b,将克隆好的pcDNA-flag-Hsf4b转染HEK293T细胞,并用抗Flag抗体进行免疫印迹分析,免疫沉淀实验和体内Pulldown实验证明Hsf4b可与MAP激酶P38结合,激酶实验结果显示P38可体外磷酸化Hsf4b。结果:应用基因克隆技术,将人Hsf4b的cDNA克隆到真核表达质粒pcDNA3.0和pEBG中。pcDNA-Flag-Hsf4b可在真核细胞HEK293T中表达一个相对分子质量(Mr)约为60000的蛋白。进一步研究发现,Hsf4b可与MAP激酶P38结合,Hsf4b的C-端转录调控区参与和P38的结合,P38可体外磷酸化Hsf4b。结论:实验首次证明Hsf4b可结合并被P38磷酸化,为进一步探讨Hsf4b在晶状体发育过程中的作用提供新的信号通路。
OBJECTIVE: To construct an eukaryotic expression vector for heat shock transcription factor 4b (Hsf4b), and to investigate the role of MAP kinase P38 in its phosphorylation. Methods: Human heart cDNA library was used as a template. PCR was carried out using a primer containing the full length of Hsf4b and a Flag tag was added to the N-terminal of Hsf4b cDNA. The PCR product was digested with KpnI and EcoRI and ligated with the digested linearized pcDNA3.0 plasmid to obtain the recombinant plasmid pcDNA-Flag-Hsf4b. The cloned pcDNA-flag-Hsf4b was transfected into HEK293T cells and transfected with anti-Flag antibody Western blot analysis, immunoprecipitation and pulldown experiments in vivo showed that Hsf4b could bind to MAP kinase P38, and kinase experiments showed that P38 could phosphorylate Hsf4b in vitro. Results: cDNA of human Hsf4b was cloned into eukaryotic expression plasmids pcDNA3.0 and pEBG by gene cloning technique. pcDNA-Flag-Hsf4b can express a protein with molecular weight (Mr) of about 60,000 in eukaryotic HEK293T cells. Further studies showed that Hsf4b could bind to MAP kinase P38, the C-terminal transcription regulatory region of Hsf4b was involved in binding to P38, and P38 could phosphorylate Hsf4b in vitro. CONCLUSION: It was first demonstrated by experiments that Hsf4b can be combined and phosphorylated by P38, providing a new signaling pathway for further exploring the role of Hsf4b in lens development.