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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 eukaryotic expression vector pcDNA3.1 (-) / NNMT (nicotinamide-N-methylase) and stably transfected it into hepatocellular carcinoma cell line SMMC7721. Methods The full length of NNMT cDNA was cloned by PCR from PGEX4T-1 / NNMT recombinant plasmid. The amplified cDNA fragment was subcloned into eukaryotic expression vector pcDNA3.1 (-) after being ligated with pMD19-T vector. The recombinants were identified by restriction enzyme analysis and sequencing. The recombinant plasmids were transfected into human hepatocellular carcinoma cell line SMMC7721 by lipofection technique. The recombinant plasmids were screened by G418 and stable transfected cell lines were established. RT-PCR was used to detect the cells before and after transfection Strain NNMT gene mRNA expression level. Results The eukaryotic expression vector was constructed successfully. The mRNA expression level of NNMT gene was higher than that of the control group by RT-PCR. The results confirmed that the NNMT gene was stably transfected into SMMC7721 cells and expressed. Conclusion The stable transfected cell line of human NNMT was successfully established, which laid the foundation for further study of the function of NNMT.