人MDM2基因真核表达载体的构建及其与p53的相互作用

目的:构建带Flag标签的MDM2真核表达载体,并检测MDM2与p53的相互作用。方法:从人乳腺文库中PCR扩增MDM2编码序列,将其插入pcDNA3.0-Flag载体,转染293T细胞后用Western印迹检测其在293T细胞中的表达,并通过免疫共沉淀实验检测MDM2与p53的相互作用。结果:双酶切和测序结果表明,Flag-MDM2真核表达载体构建成功,转染293T细胞后成功表达;免疫共沉淀实验证明Flag-MDM2与p53存在相互作用。结论:构建了带Flag标签的人MDM2真核表达载体,并检测了MDM2与p53之间的相互作用,为研究MDM2的功能奠定了基础。

Construction of Flag-Tagged Human MDM2 Eukaryotic Expression Vector and Detection of Interaction Between MDM2 and p53

Objective： To construct the eukaryotic expression vector of human MDM2 labeled with Flag tag and detect the interaction with p53. Methods： Human MDM2 gene was obtained from human mammary cDNA library by PCR and cloned into Flag vector. Human 293T cells were transfected with the recombinant plasmid of Flag-MDM2 and the expression was detected by Western blot. Interaction between MDM2 and p53 was detected by im-munoprecipitation. Results： MDM2 eukaryotic expression vector labeled with Flag tag was successfully constructed. The expression of Flag-MDM2 was identified by Western blot in human 293T cells. Flag-MDM2 interacted with p53. Conclusion： The eukaryotic expression vector of Flag-MDM2 was successfully constructed and interacted with p53, which had laid foundation for the further study of the function of MDM2.