小鼠雄激素受体真核表达载体的构建及稳定转染TM4细胞系的建立与鉴定

Construction of eukaryotic expression vector of mouse AR and establishment of its stable transfected TM4 cell line

目的构建小鼠雄激素受体（androgenreceptor，AR）基因真核表达载体，转染TM4细胞，建立稳定转染AR的TM4细胞系。方法应用RT．PCR方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，将载体以脂质体方式转染至TM4细胞，通过G418筛选稳定转染AR的TM4细胞株，以RT．PCR和Westem-blot检测AR在转染前后TM4细胞中的表达情况。结果成功构建了pcDNA3．1（-）Ag表达质粒，建立了稳定转染的TM4细胞系。RT-PCR和Western-blot检测结果表明，AR基因在该细胞系中成功表达。结论AR真核表达载体成功构建和稳定转染TM4细胞系的建立为进一步体外研究AR的功能奠定了基础。

Objective To construct eukaryotic expression vector of mouse （androgen receptor, AR） and establish its stable transfected TM4 cell Iine. Methods The AR was amplified from mouse testis by RT-PCR. The sequenced PCR products were cloned into pcDNA3.1 （-） vectors. The recombined plasmid pcDNA3.1 （-）/AR was sequenced and then transfected into TM4 cell with lipofectamineTM2000. Stable transfected TM4 celI line was established by G418 screening culture. The expression of AR was further identified by RT-PCR and Western-blot. Results The eukaryotic expression plasmid of pcDNA3.1（-）/AR was successfully constructed and stable transfected TM4 cell line was established. Stable expression of AR was detected in stable transfected TM4 cells by RT-PCR and Western-blot. Conclusion The recombinant eukaryotic expression vector ofpcDNA3. I（-）/AR and its stable transfected TM4 cell line were successfully established, providing a foundation for further function study of AR in vitro.