摘要
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。
AIM：To construct eukaryotic plasmid expressing Hsf4b and to investigate Hsf4b is phosphorylated by MAP kinase P38. METHODS：The total RNA of human heart tissues were prepared. Hsf4b cDNAs were then synthesized with RT-PCR,The PCR products were digested with Kpn I and EcoR I and sublconed into pcDNA3.0,pcDNA-Flag-Hsf4b was transfected into HEK293T cells,The expression of Hsf4b was testified with western blotting. The interaction between Hsf4b and P38 was assayed by Immunoprecipitation. In vivo pull down GST demonstrated that Hsf4b（196-493） could interact withP38,P38 phosphorylation of Hsf4b were testified with Kinase assay. RESULTS：We subcloned the human cDNA of Hsf4b into eukaryotic expression vectors pcDNA3 and PEBG,and Hsf4b was over-expressed in HEK293T cells. further studies demonstrated that Hsf4b could interact with and phosphorylated by MAP kinase P38.Hsf4b C-terminal participates the association with P38. CONCLUSION：Hsf4b could interact with and phosphorylated by MAP kinase P38.Our results will provide more evidence for understanding the signal regulation of Hsf4b transcription activity during lens development.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第4期325-328,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30871299)
