CUL4A-DDB1细胞稳定株筛选及DNA双链断裂模型构建

Establishment of CUL4A/DDB1 stable expression cell strain and cell models responding to DNA damage

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 find the dynamic change of one or more key components of the CUL4A-DDB1 complex involved in the process of DNA damage and repair and to construct tandem affinity purification（TAP） targeted plasmids of CUL4A/DDB1 and cell models responding to DNA damage. Methods The full-length gene of CUL4A/ DDB1 by PCR was used to express the recombinant expression vector pNTAP-A-CUL4A/DDB1. Exposure to eisplatin, irradiation and other cellular stresses was performed to select appropriate cell models responding to DNA damage. After the G418 resistant cells were induced, CUIAA/DDB1 activity of the supernatant was determined. Results and Conclusion The CUIAA and DDB1 genes by PCR were amplifid and then recombined into the pNTAP-A expression vector. Appropriate cell models responding to DNA damage were successfully selected arid cell lines that stably express CUL4A/DDB1 were obtained. These lines can be used formass chromatography to analyze the functional role of CUL4A-DDB1 E3 ligase in DNA damage and repair.