CAJ | 학술논문

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。
배경：범가니빈혈（ FA）통로작위DNA교련손상수복적주요통로재유지염색체은정성방면기중요작용。근년래，유관FA통로재DNA손상이급종류발병궤제등방면적연구월래월다。목적：연구인결직장암세포주SW480중RAD18단백여FANCD2단백시부존재상호작용。방법：응용면역공침정법침정항원항체복합물；응용단백질인적법검측복합물중토항인FANCD2화RAD18단백적표체。장GST-RAD18화GST질립분별전화도BL21세균중유도목적단백표체，제취세균총단백，이GST경지당주결합GST-RAD18단백，여SW480세포렬해액부육후，가입토항인FANCD2항체，진행단백질인적검측。결과：재이용FANCD2항체침정하래적복합물중가검측도RAD18단백，차재이용RAD18항체침정하래적복합물중가검측도FANCD2단백；GST-RAD18단백작유이단백시가이포획SW480세포중적FANCD2단백。결론：RAD18단백여 FANCD2단백재 SW480세포내존재상호작용；GST-RAD18단백여FANCD2단백야존재상호작용。
BacKground:Fanconi anemia( FA)pathway as a DNA crossIink damage repair pathway pIays an important roIe in maintaining genome stabiIity. In recent years,FA pathway was wideIy studied in DNA damage and cancer pathogenesis. Aims:To investigate the interaction between RAD18 and FANCD2 protein in human coIorectaI cancer ceII Iine SW480. Methods:Antigen-antibody compIex was co-precipitated by immunoprecipitation. Expressions of rabbit anti-human FANCD2 and RAD18 protein in antigen-antibody compIexes were detected by Western bIotting. The pIasmids of GST-RAD18 and GST were transferred into BL21 ceIIs and induced to express the target proteins. TotaI proteins of the ceII was extracted and GST-beads were used to conjugate the GST-RAD18 protein,and then incubated with the SW480 ceII Iysates, and Western bIotting was performed with the addition of rabbit anti-human FANCD2 antibodies. Results:RAD18 protein was detected in the antigen-antibody compIex from immunoprecipitation by using anti-FANCD2 antibody,and FANCD2 protein was detected by using anti-RAD18 antibody. FANCD2 protein was aIso detected by using anti-GST-RAD18 antibody. GST-RAD18 protein used as bait protein couId capture the FANCD2 protein in SW480 ceIIs. Conclusions:There is an interaction between RAD18 and FANCD2 protein in SW480 ceIIs,and aIso an interaction between GST-RAD18 and FANCD2 protein.