NNMT真核表达载体的构建并转染SMMC7721肝癌细胞株

The Establishment and Identification of the Stable Transfected SMMC7721 Cell Expressing NNMT

目的构建pcDNA3.1(-)/NNMT(尼克酰胺-N-甲基化酶)真核表达载体并将其稳定转染到肝癌细胞系SMMC7721中。方法采用PCR法从PGEX4T-1/NNMT重组质粒中克隆得到NNMTcDNA全长序列,并将扩增的cDNA片段与pMD19-T载体连接后亚克隆到真核表达载体pcDNA3.1(-)中。重组子经酶切分析及测序鉴定后,用脂质体转染技术将其导入到人肝癌细胞系SMMC7721,经G418筛选并建立稳定的转染细胞株,应用RT-PCR检测转染前后该细胞株NNMT基因的mRNA表达水平。结果真核表达载体构建成功,经RT-PCR检测,重组质粒转染株的NNMT基因mRNA表达水平高于对照组,证实NNMT基因已经稳定转染到SMMC7721细胞中并得到表达。结论成功建立了人基因NNMT的稳定转染细胞株,为进一步研究NNMT的功能奠定了基础。

Objective To construct a stable transfected of human hepatoma cancer cell line SMMC7721 that can highly express human NNMT. Method NNMT gene was amplified from recombinant plasmid pGEX4T-1/NNMT by PCR method, and cloned into vector pMD19-T. Then cloned into eukaryotic expression vector pcDNA3.1 （-） and the recombinant plasmid was named as pcDNA3.1-NNMT. Lipofectamine 2000 was used to transfect the pcDNA3.1-NNMT into SMMC7721 cells. The NNMT-transfected SMMC7721 cells were selected with G418 for 4 weeks. Finally, RT-PCR was used to detect the expression of human NNMT in SMMC7721 cells. Result We have successfully constructed the eukaryotic expression vector for the expression of NNMT. Conclusion This study indicated NNMT gene could be stably expressed in SMMC7721 cells and its functions on tumor cells could be further analyzed by this stable cell line.