人cystatinB基因在哺乳动物细胞株中的定位与表达

Location and expression of human cystatin B gene in mammalian cell lines

目的用基因克隆方法构建带FLAG-tag的人cystatinB真核表达载体,用其转染COS-7和HEK 293 T细胞观察cystatin B在增殖细胞内的定位;转染HEK 293 T细胞检测其表达。方法设计带FLAG-tag的引物,以人cystatin B全长cDNA序列的质粒为模板,PCR法扩增cystatin B全长序列,然后插入pCDNA 3中构建pCDNA 3-cystatin B-FLAG(CSTBF)质粒;脂质体法转染至COS-7、HEK 293 T细胞,荧光显微镜下观察其在细胞内定位;转染至HEK 293 T细胞,提取细胞总蛋白进行Western blot。结果正确构建了pCD-NA 3-cystatin B-FLAG质粒;定位实验表明在COS-7和HEK293 T细胞中,cystatin B主要分布在细胞核内,核膜处更集中,胞浆内也有广泛分布;Western blot结果表明该质粒能在细胞中有效表达。结论该研究结果为了解cystatin B在细胞内与其他蛋白质相互作用及功能提供了一定的基础。

Objective To construct FLAG-tagged human cystatin B vector with gene clone,transfect into mammalian cell lines covering COS-7 and HEK 293 T,and to investigate the location and expression of cystatin B.Methods The full length cDNA fragment of cystatin B was used to PCR amplify which promoted by a couple of FLAGtagged primers.The yield was inserted into pCDNA3 vector to construct a pCDNA3-cystatin B-FLAG（CSTBF） plasmid followed by transfection with leposomes.Images of location of the protein within COS-7 and HEK 293 T were obtained by fluorescence microscope,while expression was detected by Western blot.Results Cystatin B not only predominantly located within nucleus especially surrounding the nuclear membrane,but also within cytoplasm extensively.Furthermore,the protein was detected in the lysate of HEK 293 T cells.Conclusion The results provide a basis for interaction and functions with other protein.