人RACK1蛋白在HEK-293T和BL21细胞中的表达及COS7细胞的定位  

The expression of human RACK1 in HEK-293T and BL21 and localization in COS7

作　　者：乔正[1] 赵健[1] 潘林鑫[1] 李春雨[1] 徐雪琴[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生命科学院生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2014年第9期1189-1192,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);安徽省教育厅自然科学重点科研项目(编号:KJ2010A187)

摘　　要：目的构建可以表达有活性的蛋白激酶C受体(RACK1)的重组质粒,检测其在细胞内的表达与定位情况。方法设计上下游引物,以RACK1全长cDNA序列为模板,PCR扩增出RACK1序列,分别构建到载体pcDNA3.1和pGEX-5X-3。转染pcDNA3.1-FLAG-RACK1到人胚胎肾细胞(HEK-293T)和非洲绿猴肾成纤维细胞(COS7细胞),转化pGEX-5X-3-RACK1到大肠杆菌BL21感受态细胞中表达,Western blot法和考马斯亮蓝染色法检测RACK1表达情况,免疫荧光法研究其细胞内定位。结果成功构建了pcDNA3.1-FLAG-RACK1和GEX-5X-3-RACK1重组质粒。Western blot证明了其在HEK-293T细胞中的表达,考马斯亮蓝染色显示其在BL21菌株也能大量表达,免疫荧光显示RACK1在COS7的细胞核与细胞质中均有分布。结论人RACK1在HEK-293T细胞和BL21菌株中均能有效表达,在COS7细胞的胞质、胞核均有分布,为进一步研究人RACK1基因的功能奠定了基础。Objective To construct an available recombinant plasmids of RACK1 and detect its expression and lo- calization in ceils. Methods The upstream and downstream primers of RACK1 were designed, RACK1 was ampli- fied by PCR with the template containing its full length cDNA fragment, and then the eukaryotic plasmid pcD- NA3.1-FLAG-RACK1 and the protokaryotic plasmid pGEX-SX-3-RACK1 were constructed, pcDNA3.1-FLAG- RACKl was transfected into HEK-293T and COS7 cells, and pGEX-SX-3-RACK1 was transformed into BL21 cells. Western blot and Coomassie brilliant blue staining were used for the expression and immunofluorescence used for the localization. Results The recombinant plasmids pcDNA3.1-FLAG-RACK1 and pGEX-SX-3-RACK1 were constructed successfully. RACK1 can be expressed in HEK-293T cells and BL21 competent cells, and the expres- sion of RACK1 was found both in nucleus and in cytoplasms. Conclusion The protein RACK1 can be expressed effectively in HEK-293T ceils and BI221 cells and locates both in nucleus and in cytoplasm in COS7 cells, which is basic for further study of human RACK1.

关 键 词：RACK1 基因表达 细胞定位 BL21 

分 类 号：R341[医药卫生—基础医学] R394.2