乙酰胆碱受体四聚体前体α亚单位在人胚肾293细胞膜上的表达  

Expression of acetylcholine receptor tetramer precursor α subunit on the membrane of HEK293 cells

作　　者：王思远[1] 张辛轲 孙昌元[1] 李芳芳[1] 金权鑫[1] 李英信[1] 李红花[1] 孟繁平[1] 

机构地区：[1]延边大学基础医学院免疫学与病原生物学教研部,吉林延吉133002

出　　处：《延边大学医学学报》2011年第3期157-160,共4页Journal of Medical Science Yanbian University

摘　　要：[目的]将乙酰胆碱受体四聚体前体!亚单位重组质粒(pcDNA3.1-AchRα-BirA)转染至人胚肾293(HEK293)细胞,并在其细胞膜上获得稳定表达.[方法]提取重组质粒pcDNA3.1-AchRα-BirA,经限制性核酸内切酶Eco RⅤ单酶切后,行低熔点琼脂糖凝胶电泳检测.采用脂质体转染法,将pcDNA3.1-AchRα-BirA转染至HEK293细胞,经抗生素G418筛选后形成集落状抗性克隆细胞.将表达产物扩大培养,应用免疫荧光技术,以异硫氰酸荧光素标记,经荧光显微镜查看表达情况.[结果]电泳检查发现了大小约为6.964 kb的条带.转染后G418筛选获得抗性克隆细胞,经荧光显微镜观察到HEK293细胞膜上的绿色荧光.[结论]成功地将AchRα-BirA基因转染至HEK293细胞膜上且有表达,为下一步构建AchRα四聚体奠定了基础.OBJECTIVE To express the recombinant vector bearing acetylcholine receptor （AchR） α subunit （pcDNA3.1-AchRa-BirA） on the membrane of human embryonic kidney 293 （HEK293） cell line. METHODS The recombinant plasmid vector was obtained from the E. coli DH5a, digested with restriction endonuclease Eco R V, and then analyzed by low-melting agarose gel electrophoresis. Furthermore the recombinant plasmid pcDNA3.1-AchRα-BirA was transfected into HEK293 cell by the liposome-mediated method. The clones with high expression were selected by antibiotics G418. The gene expression of pcDNA3.1-AchRα-BirA on the membrane of HEK293 cells was analyzed by immunofluorescence technology. RESULTS A 6. 964 kb target fragment was observed in agarose gel electrophoresis. After transfection, several expression clones were obtained with the selection of G418. The green fluorescence on the membrane of HEK293 was observed by fluorescence microscopy. CONCLUSION The recombinant vector pcDNA3.1-AchRα-BirA recognition polypeptide sequence gene is successfully expressed on the membrane of HEK293 cells, and this will lay a foundation for the construction of AchRα tetramer.

关 键 词：受体 胆碱能 HEK293细胞 脂质体转染 

分 类 号：R392.12[医药卫生—免疫学]