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

摘要

目的 构建小鼠雄激素受体(androgen receptor,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Western-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(-)/AR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 line. 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 cell 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. Stabfe expression of AR was detected in stable transfected TM4 cells by RT-PCR and Western-blot. Conclusion The recombinant eukaryotic expression vector of pcDNA3.1(-)/ARand its stable transfected TM4 cell line were successfully established, providing a foundation for further function study of AR in vitro.