脆性组氨酸三联体与复制蛋白A相互作用的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR／CHKl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。

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

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.