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目的:构建pcDNA3.1(-)/VCC-1真核表达载体并将其稳定转染到肝癌细胞系SMMC-7721中。方法:以SMMC-7721细胞总RNA为模板,通过RT-PCR扩增VCC-1基因编码区cDNA,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SMMC-7721,经G418筛选并建立稳定的转染细胞株,应用RT-PCR及Western blot检测转染前后该细胞株VCC-1基因的表达。结果:pcDNA3.1(-)/VCC-1经酶切鉴定及DNA测序证实序列完全正确,真核表达载体构建成功;经RT-PCR及West-ern blot检测,重组质粒转染株的VCC-1基因的表达水平高于对照组,证实VCC-1基因稳定转染到SMMC-7721细胞中并得到表达。结论:成功地建立了人基因VCC-1的肝癌细胞SMMC-7721稳定转染株,为进一步研究VCC-1的功能奠定了基础。
Objective: To construct pcDNA3.1(-)/VCC-1 eukaryotic expression vector and stably transfect it into hepatoma cell line SMMC-7721. METHODS: The cDNA of the coding region of VCC-1 gene was amplified by RT-PCR using total RNA of SMMC-7721 cells as a template, and the amplified cDNA fragments were ligated with pMD19-T vector and subcloned into the eukaryotic expression vector pcDNA3.1. (-)in. The recombinants were identified by restriction analysis and sequencing, and then introduced into the human hepatocellular carcinoma cell line SMMC-7721 with liposome transfection technology. The stable transfected cell line was established by G418 selection and detected by RT-PCR and Western blot. The expression of the VCC-1 gene in the cell line before and after transfection. RESULTS: The sequence of pcDNA3.1(-)/VCC-1 was confirmed by restriction analysis and DNA sequencing. The sequence was completely correct and the eukaryotic expression vector was constructed successfully. The RT-PCR and West-ern blot detection showed that the recombinant plasmid was transfected with VCC- 1 The expression level of the gene was higher than that of the control group, and it was confirmed that the VCC-1 gene was stably transfected into SMMC-7721 cells and expressed. Conclusion: The stable transfection of human hepatoma cell line SMMC-7721 with human gene VCC-1 was successfully established, which laid a foundation for further study on the function of VCC-1.