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目的为了鉴定并验证CUL4A-DDB1泛素连接酶复合体中参与DNA损伤修复反应过程的1～2种关键成分在损伤识别、早期及晚期修复中的动态变化,拟构建含有串联亲和纯化(TAP)标签载体并筛选高表达CUL4A/DDB1的细胞株,建立DNA双链断裂模型。方法利用PCR获得CUL4A/DDB1基因,构建重组表达载体pNTAP-A-CUL4A/DDB1;使用顺铂和电离辐射刺激等外界刺激建立合适的DNA双链断裂细胞模型,使用G418筛选稳定表达CUL4A/DDB1的细胞株。结果与结论成功构建pNTAP-A-CUL4A/DDB1表达载体,建立合适的DNA双链断裂细胞模型,筛选得到稳定表达CUL4A/DDB1的细胞稳定株,为下一步质谱分析CUL4A-DDB1泛素连接酶在DNA损伤修复过程中的新功能打下基础。
Objective To identify and validate the dynamic changes of one or two key components involved in DNA damage repair reaction in CUL4A-DDB1 ubiquitin ligase complex during injury identification, early and late repair, ) Tag vector and screening high expression of CUL4A / DDB1 cell lines, the establishment of DNA double-strand break model. Methods The CUL4A / DDB1 gene was cloned by PCR. The recombinant plasmid pNTAP-A-CUL4A / DDB1 was constructed. The DNA double-strand breaks were induced by external stimuli such as cisplatin and ionizing radiation. Cell lines. RESULTS AND CONCLUSION: The pNTAP-A-CUL4A / DDB1 expression vector was successfully constructed and a suitable double-stranded DNA cleavage cell model was established. CUL4A / DDB1 stably expressing cells were screened out for further screening of CUL4A-DDB1 ubiquitin ligase DNA damage repair process laid the foundation for new features.