利用免疫共沉淀验证抗增殖蛋白与HSP70的相互作用

IDENTIFICATION OF INTERACTION BETWEEN HSP70 AND PROHIBITIN BY CO-IMMUNOPRECIPITATION

目的利用免疫共沉淀技术验证抗增殖蛋白(PHB)与HSP70间的相互作用,为研究应激时HSP70促进PHB进入线粒体的机制提供科学依据。方法采用Western blot方法检测HEK293细胞中PHB与HSP70的表达。构建抗增殖蛋白真核表达载体pEGFP-N1-PHB,经酶切鉴定正确后,将其转染HEK293细胞,采用荧光倒置显微镜观察转染效率。收集转染的HEK293细胞,利用免疫共沉淀技术验证PHB与HSP70之间的相互作用。结果在HEK293细胞中,HSP70具有较高的表达量,而PHB的表达量非常低。构建了抗增殖蛋白真核表达载体pEGFP-N1-PHB,转染HEK293细胞的效率达到90%以上。在抗体沉淀的PHB相互作用蛋白复合物中,可以检测到HSP70的存在。结论构建了抗增殖蛋白融合蛋白真核表达重组载体,在HEK293细胞中高表达抗增殖蛋白后,利用免疫共沉淀技术证实PHB与HSP70之间存在相互作用。

Objective To explore the interaction between HSP70 and prohibitin（PHB） by co-immunoprecipitation in order to provide scientific basis for studying the mechanism of promoting PHB into the mitochondria by HSP70 during stress.Methods The expression of prohibitin and HSP70 in HEK293 cells were assayed by Western blot method.The eukaryotic expression vector of prohibitin was constructed,identified,then HEK293 cells were transfected with it.The transfection efficiency was evaluated by inverted fluorescence microscope,and HEK293 cells were collected after transfection for 36 h.The interaction between HSP70 and prohibitin was detected by co-immunoprecipitation.Results The basic expression of HSP70 in HEK293 cells was much higher than that of prohibitin.Double restriction enzyme digestion showed that pEGFP-N1-PHB was constructed correctly.More than 90% of the cells were transfected.When prohibitin was immunoprecipitated by anti-PHB polyclonal antibody,HSP70 was identified from immunoprecipitated complex.Conclusion The recombinant vector pEGFP-N1-PHB was constructed successfully.When prohibitin was highly expressed in HEK293 cells,the interaction between HSP70 and prohibitin was confirmed by co-immunoprecipitation.