脆性组氨酸三联体与复制蛋白A相互作用的鉴定  

Identification of the Interaction Between Fragile Histidine Triad and Replication Protein A

作　　者：于芳[1] 绳纪坡[1] 张勇[1] 张鸿声[1] 高宁[1] 张宏达[1] 胡宝成[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2008年第3期324-327,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30770651;30670616;30370441);国家"十一五"支撑项目(2006BAI23B02);国家重点基础研究发展计划项目(2005CB522506;2007CB914604)

摘　　要：目的:研究脆性组氨酸三联体(Fhit)对ATR/CHK1通路的影响,确定Fhit与复制蛋白A(RPA)存在相互作用,为进一步研究Fhit特异的信号通路奠定基础。方法:将Fhit全长基因插入含GST基因的原核表达载体中,在大肠杆菌中表达纯化GST-Fhit融合蛋白,并用GST沉降技术研究Fhit与RPA是否在体外存在相互作用;在表达Fhit的人细胞中用免疫共沉淀技术分析Fhit与RPA是否在体内存在相互作用,同时用免疫荧光染色方法研究Fhit与RPA在细胞内是否可以共定位。结果:通过免疫共沉淀、免疫荧光染色及GST沉降技术,确定了Fhit与RPA在体内及体外均可以相互作用。结论:确定了Fhit与RPA之间存在相互作用,为阐明Fhit在维持基因组完整性方面的机理提供了线索。Objective： To study how fragile histidine triad（Fhit） affects the ATR/CHK1 pathway, the interaction between Fhit and replication protein A （RPA） will be determined, so as to lay a foundation for studying the specific Fhit signal pathways. Methods： Fhit full length gene was inserted into the vector containing GST gene, GST-Fhit fusion protein were expressed and purified in E.coli, the interaction between Fhit and RPA was analyzed by GST-pull down in vitro. The interaction was determined by co-immunoprecipitation in human cells expressing Fhit, and the co-localizations were analyzed by immunofluorescence staining. Results： The interaction between Fhit and RPA was determined in vivo and in vitro by using co-immunoprecipitation, immunofluorescence staining and GST-pull down assays. Conclusion： The interaction between Fhit and RPA was determined, it provides a clue to elucidate the mechanism for Fhit maintaining genomic integrity.

关 键 词：脆性组氨酸三联体 复制蛋白A 蛋白质相互作用 

分 类 号：Q25[生物学—细胞生物学] Q75