CAJ | 학술논문

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 pcD-NA3.1-FLAG-RACK1和GEX-5X-3-RACK1重组质粒。 West-ern 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。
목적구건가이표체유활성적단백격매 C 수체(RACK1)적중조질립，검측기재세포내적표체여정위정황。방법설계상하유인물，이RACK1전장cDNA서렬위모판， PCR확증출 RACK1서렬，분별구건도재체 pcDNA3.1화pGEX-5X-3。전염pcDNA3.1-FLAG-RACK1도인배태신세포(HEK-293T)화비주록후신성섬유세포(COS7세포)，전화pGEX-5X-3-RACK1도대장간균 BL21감수태세포중표체， Western blot법화고마사량람염색법검측RACK1표체정황，면역형광법연구기세포내정위。결과성공구건료 pcD-NA3.1-FLAG-RACK1화GEX-5X-3-RACK1중조질립。 West-ern blot증명료기재HEK-293T세포중적표체，고마사량람염색현시기재BL21균주야능대량표체，면역형광현시RACK1재COS7적세포핵여세포질중균유분포。결론인RACK1재HEK-293T세포화BL21균주중균능유효표체，재COS7세포적포질、포핵균유분포，위진일보연구인RACK1기인적공능전정료기출。
Objective To construct an available recombinant plasmids of RACK1 and detect its expression and lo-calization in cells. 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-5X-3-RACK1 were constructed. pcDNA3.1-FLAG-RACK1 was transfected into HEK-293T and COS7 cells, and pGEX-5X-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-5X-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 cells and BL21 cells and locates both in nucleus and in cytoplasm in COS7 cells, which is basic for further study of human RACK1 .