Smad家族蛋白在真核细胞中的表达  

Cloning and expression of Smad proteins in 293T cells

作　　者：熊志红[1] 杨丽萍[2] 李国利[1] 庄玉辉[1] 

机构地区：[1]解放军总医院第二附属医院结核病研究所,北京市100091 [2]解放军总医院第一附属医院,北京市100097

出　　处：《实用医学杂志》2009年第7期1027-1029,共3页The Journal of Practical Medicine

摘　　要：目的:探索Smad蛋白在肺部肿瘤形成过程中的分子机制,构建带FLAG标签的smad1、2、3、4的真核表达载体,并检测其在293T细胞中的表达。方法:以人肺细胞cDNA文库为模板,分别扩增smad1、2、3、4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,Western-blot法检测细胞中的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-smad真核表达载体,并都能在真核细胞中表达分子量大小相符的重组蛋白。结论:成功地构建了FLAG-smad1、2、3、4真核表达载体,为Samd蛋白及其相关蛋白在肺部肿瘤的作用研究奠定了基础。Objective To explore the molecular mechanisms of the Smad proteins in lung cancer development, we constructed the Smad proteins eukaryotic expression vectors and detected their expressions in the SV40-transformed embryonic kidney 293T cell line. Methods Conventional PCR was used to amplify the full-length coding sequences of the four Smad genes, smad-1,2,3,4. The amplified gene fragments were cloned into the eukaryotic expression vector pcDNA3-FLAG. The recombinant plasmids were transfected into the 293T cells using lipofectamine reagent, and the expression of the Smad proteins in the transfected 293T cells was detected by Western blotting assay. Results The results of restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmids were correctly constructed. The expression of the recombinant proteins was also identified by Western blotting assay. Conclusion The recombinant Smads proteins were successfully expressed in the 293T cells, which laid a foundation for further study of Smads and their interaction proteins in lung cancer development.

关 键 词：真核细胞 SMAD蛋白 克隆 

分 类 号：R734.2[医药卫生—肿瘤]