一种基于三顺反子表达载体的生物素诱导表达系统的建立

目的:建立一种以无毒的生物素为诱导剂的哺乳动物细胞诱导表达系统。方法:通过基因组PCR或重叠延伸PCR获得该系统的三个基本构件:大肠杆菌生物素连接酶(BirA)、链亲和素-四环素依赖的抑制因子融合蛋白(SA-TetR)和生物素化信号-VP16转录激活结构域融合蛋白(Avitag-VP16)。将上述基因连入三顺反子表达载体,与响应载体一起共转染293f细胞,以EGFP为报告基因,检测EGFP荧光强度随培养体系中生物素浓度变化的情况。结果:随着生物素浓度的增加,目的基因表达出现OFF-ON-OFF的变化,诱导状态下的EGFP荧光强度约为抑制状态下的3倍。结论:可通过调节生物素浓度对目的基因的表达进行可逆的调节,该系统是一种有应用前景的诱导表达系统。

Establishment of a Biotin-inducible Expression System Based on Tricistronic Expression Vector

Objective: To establish a mammalian inducible expression system with the non-toxic biotin as an inducer.Methods: This system comprised three key components: E.coli biotin ligase(BirA),streptavidin-tetracyclin repressor(SA-TetR) and biotinylation signal-VP16 transactivation domain(AT-VP16).The three genes were introduced into a tricistronic expression vector,and co-transfected 293f cells together with the response vector.The EGFP taken for a reporter gene,EGFP fluorescent intensities were detected under diff...