LHR真核表达载体构建及表达

Construction of Eukaryotic Expression Recombinant Plasmid pcDNA3.1(+)-LHR

目的构建黄体生成素受体(LHR)真核表达质粒载体和建立稳定高表达LHR的乳腺癌细胞株。方法KpnⅠ/HpaⅠ双酶切LHR-cDNA质粒载体获得目的基因,定向克隆构建pcDNA3.1(+)真核表达载体,酶切图谱和序列分析鉴定;重组质粒载体经脂质体转染MCF-7乳腺癌细胞,G418筛选,RT-PCR、细胞内cAMP测定,筛选鉴定高表达LHRMCF-7细胞。结果重组质粒pcDNA3.1(+)-LHR酶切图谱分析结果、序列分析证明pcDNA3.1(+)-LHR载体中LHR序列与GenBank的LHR基因序列完全相符。RT-PCR检测MCF-7细胞LHR mRNA,MCF-7细胞内cAMP浓度测定,证实MCF-7细胞高表达LHR。结论构建并转染pcDNA3.1(+)-LHR真核表达载体,在MCF-7细胞中稳定表达,为探讨hCG对乳腺癌细胞的作用提供实验基础。

Objective To construct the eukaryotic expression recombinant plasmid pcDNA3. （＋）-LHR and to establish human breast cancer MCF-7 cell lines stably expressing exogenous LHR. Methods LHR eDNA was cloned into pcDNA3.1（＋） vector, the recombinant plasmid pcDNA3. I（＋）- LHR was identified by restriction enzyme digestion and DNA sequence analysis, pcDNA3.1 （＋）-LHR plasmid was transfected into MCF-7 cells and cell lines stably expressing exogenous LHR were established by G418 screening. The expression of LHR was detected by RT-PCR and cAMP level analysis. Results The successful construction of pcDNA3. I（＋）-LHR was confirmed by restriction enzyme digestion and DNA sequencing analysis. RT-PCR and cellular cAMP level assay confirmed the stable expression of exogenous LHR in MCF-7 cells. Conclusion MCF-7 cell line stably expressing exogenous LHR could provide a useful model for studying the function of hCG in human breast cancer cells.