摘要
目的构建带有FLAG-tag的人野生型EB病毒诱导基因3(EBI3)真核表达载体,用其转染COS-7和HEK 293T细胞,观察EBI3在哺乳动物细胞中的定位和表达情况。方法设计带有FLAG-tag的引物,以含人EBI3全长cDNA序列的质粒为模板,以PCR方法扩增出EBI3全长序列,然后插入真核表达载体pCDNA3.1中,构建pCDNA3.1-EBI3-FLAG质粒。将重组质粒转染至COS-7细胞,荧光显微镜下观察其在胞内的定位情况;转染至HEK 293T细胞,提取细胞总蛋白进行Western blot。结果正确构建pCDNA3.1-EBI3-FLAG质粒;荧光定位实验表明在COS-7细胞中,EBI3蛋白主要分布在细胞质中,且较集中分布于靠近核膜的位置。Western blot结果表明该质粒能在细胞中有效表达。结论 EBI3在COS-7细胞中有表达,且主要分布在胞质内,该研究结果为了解EBI3在细胞内与其他蛋白质相互作用及功能奠定了一定基础。
Objective To construct FLAG-tagged EBI3 vector with gene clone transfect into mammalian cell lines COS-7 and HEK 293T, and to investigate the location and expression of EBI3. Methods The full length cDNA fragment of EBI3 was amplified by PCR, after having been promoted by a couple of FLAG-tagged primers. The yield was inserted into pCDNA3.1 vector to construct a pCDNA3.1-EBI3-FLAG plasmid, which was then followed by transfection. Location of the protein within COS-7 was obtained by fluorescence microscope, while expression was detected by Western blot. Results pCDNA3.1-EBI3-FLAG was constructed successfully. EBI3 was confirmed to be predominantly located within cytoplasm, especially surrounding the nuclear membrane by immunofluorescence. Furthermore, the protein was detected in the lysate by Western blot. Conclusion The results provided a glimpse into EB13＇ s interaction and functions with other protein.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第4期333-336,共4页
Acta Universitatis Medicinalis Anhui
基金
安徽省教育厅自然科学重点科研项目(编号:KJ2010A187)
安徽省自然科学基金(编号:11040606M170
11040606M164)
