人AK3基因在哺乳动物细胞系中的表达与定位  

Expression and localization of transfected AK3 gene in mammalian cell lines

作　　者：李克娟[1] 顾彧[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生命科学学院生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2014年第11期1531-1534,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年基金(编号:81201368);安徽省自然科学基金(编号:11040606M170;11040606M164);安徽医科大学博士科研资助项目(编号:XJ201009)

摘　　要：目的运用基因克隆技术构建带FLAG标签的腺苷酸激酶3(AK3)真核表达载体,观察其在哺乳动物细胞内的定位和表达情况。方法以人AK3全长的cDNA序列的菌液为模板,设计带有FLAG标签的引物,用PCR方法构建出真核表达载体pcDNA3.1-AK3-FLAG。运用免疫荧光及Western blot法检测其在哺乳动物细胞中的定位与表达情况。结果成功构建了pcDNA3.1-AK3-FLAG质粒;荧光定位试验表明AK3在COS7细胞的胞质中呈斑点状分布,细胞核中未见明显分布;Western blot法检测表明AK3在HEK-293T细胞中能有效表达。结论 AK3在哺乳动物细胞内能够有效表达和定位,为进一步研究AK3和其他蛋白之间的相互作用奠定了基础。Objective To construct FLAG-tagged human the localization and expression of human AK3. Methods eukaryotic expression vector of human AK3, investigate Bacteria liquid containing the full-length DNA sequence of AK3 was used as template for PCR to construct eukaryotic expression plasmid pcDNA3.1-AK3-FLAG. The local- ization and expression of AK3 in mammalian cells were detected by immunofluorescence and Western blot assay. Results The recombinant plasmid pcDNA3. 1-AK3-FLAG was constructed successfully; the human AK3 protein was found to localize mainly in cytoplasm, not in the nucleus obviously in COS7 cells;the expression vector could express AK3-FLAG efficiently in HEK-293T cells by Western blot assay. Conclusion The expression vector can express AK3-FLAG efficiently in mammalian cells, and the results are basic for further study on the function and in- teraction partners of AK3 protein.

关 键 词：AK3 细胞定位 免疫荧光 

分 类 号：R341[医药卫生—基础医学] R394.2