雌激素受体β转录激活系统的构建

Establishment of Estrogen Eeceptor β Transcriptional Activation System

目的:构建雌激素受体β(ERβ)的转录激活系统。方法:以pcDNA3-ERβ质粒为模板,利用PCR技术扩增ERβ全长及转录激活结构域1(AF1)、DNA结合结构域(DBD)、转录激活结构域2(AF2)等不同长度的基因片段,分别插入pGAL载体中,构建重组质粒,转染293T细胞,利用免疫杂交方法鉴定其表达情况,用萤光素酶报告基因(Gal4-LUC)检测转录活性。结果:构建了ERβ全长及不同功能区片段编码基因的重组质粒,转染293T细胞后检测到相应蛋白的表达;在活性实验中,雌激素(E2)诱导下pGAL-ERβ使Gal4-LUC活性升高约17倍,pGAL-ERβAF1在有无E2诱导下均能使Gal4-LUC活性升高2倍,pGAL-ERβAF2在E2诱导下使Gal4-LUC活性升高约7倍,pGAL-ERβDBD对Gal4-LUC活性没有明显作用。结论:ERβ的转录激活系统构建成功。

Objective： To establish estrogen receptor β（ERβ） transcriptional activation system. Methods： The coding sequences of full length ERβ and its different function regions including transcriptional activity function domain AF1, AF2 and DNA binding domain（DBD） were amplified from pcDNA3-ERβ by PCR and cloned into the pGAL vector. We detected the aimed proteins after the recombined plasmids were transfected into 293T cells by Western blot. The transcriptional activity of full length ERβ and its different function regions were detected in 293T cells by Gal4-LUC reporter. Results： The full length ERβ and its different function regions were expressed in the 293T cells. Compared to the empty pGAL vector, the transcription activities of full length ERβ and AF2 were induced approximately 17 and 7 fold respectively in the presence of estrogen E2 in 293T ceils, and the transcription activity of the AF1 was induced approximately 2 fold with and without E2, however, there was almost no change of DBD transcriptional activity. Conclusions： The transcriptional activity system of estrogen receptor β was successfully established.