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[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] The purpose of this study was to transfect the recombinant adenovirus vector (pcDNA3.1-AchRα-BirA) into human embryonic kidney 293 (HEK293) cells and obtain stable expression on its cell membrane. [Methods] The recombinant plasmid pcDNA3.1-AchRα-BirA was digested with EcoRV restriction endonuclease and then detected by low melting point agarose gel electrophoresis.The pcDNA3.1-AchRα- BirA was transfected into HEK293 cells and screened by antibiotic G418 to form colony-resistant cloned cells.Expression of the product was expanded and cultured.Immunofluorescence technique was used to label fluorescein isothiocyanate and the expression was observed by fluorescence microscopy. [Results] The band of about 6.964 kb was found by electrophoresis.The resistant clone cells were screened by G418 after transfection, and the green fluorescence of HEK293 cell membrane was observed by fluorescence microscope. [Conclusion] The AchRα-BirA gene was successfully transfected into HEK293 Cell membrane and expression, laid the foundation for the next step to construct AchRα tetramer.