HSP90高表达细胞系的建立及其对底物蛋白结合能力的影响

Establishment of HSP90 Overexpressing Cell Line and Analysis of the Binding Ability of HSP90 with Substrate Proteins

目的建立HSP90稳定高表达细胞系,探讨HSP90在应激适应中的作用及其机制。方法含人类HSP90β全长基因的质粒pSmycHSP经酶切鉴定、大肠杆菌转化、质粒提取纯化后,以电穿孔转染,G418筛选建立HSP90稳定高表达的小鼠成纤维细胞系。通过免疫共沉淀方法,观察HSP90表达与底物蛋白HSP70、Raf-1的结合情况。结果经G418筛选的阳性克隆HSP90clone细胞HSP90胞膜、胞核强染。免疫共沉淀结果表明,细胞内HSP90含量、HSP90结合的Raf-1量、HSP90结合的HSP70量变化趋势为外源性HSP90转染细胞>对照。结论HSP90高表达导致HSP90蛋白与部分底物蛋白的结合性改变。提示细胞可通过HSP90分子伴侣功能,抗衡应激反应中蛋白质变性引起的细胞损伤。

Objective To explore the function and mechanism of HSP90 in stress adaptation by construct HSP90 highly expressing cell lines. Method The recombinant plasimid pSmycHSP, which contains the full length cDNA of human HSP90β,was introduced into mouse fibroblast cell line NIH-3T3 by electroporation. The transfected cells were screened by G418; the positive clones were selected and identified by immunofluorescence and Western- blotting. HSP90 binding abilities with suhstrate protein HSP70, Raf-1 were observed by HSP90 coimmunoprecipitation. Result The increased level of HSP90 in transfected cell line was shown by immunofluorescence and Westen- blotting. The binding ability of HSP90 with HSP70 and Raf-1 in the transfect ceils was better than the control. Conclusion The NIH-3T3 derived cell line, which stably expressed high level of HSP90, was established. The high- level HSP90 expressed in ceils may show protection under severe circumstance, through HSP90 relocation and protecting the vital proteins in stress signal transduction pathway.