细胞周期蛋白B1、D1和E真核表达载体的构建及表达

目的：为研究细胞周期蛋白（cyclin）在肿瘤形成过程中的分子机制，构建带FLAG标签的细胞周期蛋白B1、D1、E的真核表达载体，并检测其在293T细胞中的表达。方法：以乳腺cDNA文库为模板，分别扩增细胞周期蛋白B1、D1、E基因全长编码区序列，克隆到pcDNA3-FLAG真核表达载体上；用脂质体介导的基因瞬时转染法，将重组正确的表达载体转染293T细胞，检测细胞中的FLAG融合蛋白的表达。结果：酶切鉴定和DNA序列分析显示构建了正确的FLAG-Cyclin真核表达载体，Western印迹分析表明克隆的载体都能在真核细胞中表达分子大小相符的重组蛋白。结论：构建了FLAG-CyclinBl、FIAG-CyclinDl、FLAG-CyclinE真核表达载体，为细胞周期蛋白及其相关蛋白的研究奠定了基础。

Cloning and Eukaryotic Expression of Cyclin B1,D1,E1

Objective： In order to explore the molecular mechanisms of cyclins in cancer development, the cyclin B1, DI and E eukaryotic expression vectors with FLAG epitope were construct and their expression in SV40-transformed embryonic kidney cell line 293T was detected. Methods： Standard PCR was used to amplify the full-length coding sequence of the three cyclins genes. The amplified genes were cloned into the eukaryotic expression vector pcDNA3-FLAG, generating FLAG-cyclins. The expression in the transient transfected 293T cells was detected by Western blot. Results： The result of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct. The expression of the recombinant proteins was shown by Western blot. Conclusion： The recombinant cyclins were successfully expressed, which laid foundation for further study of cyclins and their interaction proteins.