【摘要】 背景:范可尼贫血(FA)通路作为DNA交联损伤修复的主要通路在维持染色体稳定性方面起重要作用。近年来,有关FA通路在DNA损伤以及肿瘤发病机制等方面的研究越来越多。目的:研究人结直肠癌细胞株SW480中RAD18蛋白与FANCD2蛋白是否存在相互作用。方法:应用免疫共沉淀法沉淀抗原抗体复合物;应用蛋白质印迹法检测复合物中兔抗人FANCD2和RAD18蛋白的表达。将GST-RAD18和GST质粒分别转化到BL21细菌中诱导目的蛋白表达,提取细菌总蛋白,以GST琼脂糖珠结合GST-RAD18蛋白,与SW480细胞裂解液孵育后,加入兔抗人FANCD2抗体,进行蛋白质印迹检测。结果:在利用FANCD2抗体沉淀下来的复合物中可检测到RAD18蛋白,且在利用RAD18抗体沉淀下来的复合物中可检测到FANCD2蛋白;GST-RAD18蛋白作诱饵蛋白时可以捕获SW480细胞中的FANCD2蛋白。结论:RAD18蛋白与FANCD2蛋白在SW480细胞内存在相互作用;GST-RAD18蛋白与FANCD2蛋白也存在相互作用。更多还原

【Abstract】 Background: Fanconi anemia( FA) pathway as a DNA crosslink damage repair pathway plays an important role in maintaining genome stability. In recent years,FA pathway was widely studied in DNA damage and cancer pathogenesis.Aims: To investigate the interaction between RAD18 and FANCD2 protein in human colorectal cancer cell line SW480.Methods: Antigen-antibody complex was co-precipitated by immunoprecipitation. Expressions of rabbit anti-human FANCD2 and RAD18 protein in antigen-antibody complexes were detected by Western blotting. The plasmids of GSTRAD18 and GST were transferred into BL21 cells and induced to express the target proteins. Total proteins of the cell was extracted and GST-beads were used to conjugate the GST-RAD18 protein,and then incubated with the SW480 cell lysates,and Western blotting was performed with the addition of rabbit anti-human FANCD2 antibodies. Results: RAD18 protein was detected in the antigen-antibody complex from immunoprecipitation by using anti-FANCD2 antibody,and FANCD2 protein was detected by using anti-RAD18 antibody. FANCD2 protein was also detected by using anti-GST-RAD18 antibody. GST-RAD18 protein used as bait protein could capture the FANCD2 protein in SW480 cells. Conclusions:There is an interaction between RAD18 and FANCD2 protein in SW480 cells,and also an interaction between GST-RAD18 and FANCD2 protein.更多还原