小鼠雄激素受体真核表达载体的构建与鉴定

Construction and Identification of Eukaryotic Expression Vector of AR in TM4 Cell Line

目的:克隆小鼠雄激素受体(androgen receptor,AR)基因的cDNA,构建AR真核表达载体,并在睾丸支持细胞TM4中进行表达和鉴定。方法:应用RT-PCR法从小鼠睾丸中扩增AR,将测序正确的PCR产物克隆至pcDNA3.1(-)真核表达载体中,再将载体以脂质体方式转染至TM4细胞系中,通过G418筛选稳定转染AR的TM4细胞株,以RT-PCR法和Western blotting法检测转染前后AR在TM4细胞系中的表达情况。结果:RT-PCR法和Western blotting法检测的结果显示,转染pcDNA3.1(-)/AR质粒的细胞中AR基因的表达水平显著高于转染pcDNA3.1(-)质粒的对照组。结论:成功构建了小鼠AR真核表达载体pcDNA3.1(-)/AR和稳定转染的TM4细胞系,为进一步研究AR在睾丸支持细胞中的作用及其分子机制建立了细胞模型。

Objective： To clone the cDNA of mouse androgen receptor（AR） gene,and to construct eukaryotic expression vector of AR for expression and identification in TM4 celI line.Methods： The AR was amplified from mouse testis by RT-PCR.The sequenced PCR products were cloned into pcDNA3.1（-） vector.The recombined plasmid pcDNA3.1（-）/AR was sequenced and transfected into TM4 cell by lipofectamineTM 2000.After screening culture by G418,stable transfected TM4 cell line was established,the expression of AR was identified by RT-PCR and Western blotting.Results： The results of RT-PCR and Western blotting showed the expression level of AR gene was increased in stable transfected TM4 cell line.Conclusion： The recombinant eukaryotic expression vector of pcDNA3.1（-）/AR has been constructed successfully and stably expressed in TM4 cell line,which establishing a cell model to study the function and molecular mechanism of AR in Sertoli cells.