IDO基因稳定转染HepG_2细胞系的建立

Construction of a HepG_2 Cell Line with IDO Gene Steady Transfection

目的建立稳定转染IDO基因的HepG2细胞系,为进一步研究IDO基因在肝癌免疫逃逸中的作用奠定基础。方法应用脂质体将真核细胞表达质粒pcDNA3.1-IDO和空载质粒pcDNA3.1转染入HepG2细胞,经G418进行筛选后,挑取单克隆进行培养,用反转录酶聚合酶链反应(RT-PCR)和Western blot方法验证获得的表达细胞株。结果经RT-PCR和Western blotting检测证实空质粒转染组和空白对照组HepG2细胞无IDO基因及蛋白的表达,而重组质粒组的HepG2细胞表达IDO基因和蛋白。结论 pcDNA3.1-IDO质粒体外稳定转染人肝癌细胞HepG2,可以有效地使IDO基因在RNA水平和蛋白水平均表达,成功建立了稳定转染基因IDO的HepG2细胞系,为进一步探讨该基因的功能奠定了一定基础。

Objective To establish a human hepatoma carcinoma cell line with IDO gene steady transfection and verify the gene expression of IDO in HepG2 cell,which provides the foundation for the tumorous immune escape effect of pcDNA3.1-IDO.Methods The eukaryotic expression plasmid pcDNA3.1-IDO and empty vector pcDNA3.1 were transfected into HepG2 cells with lipofectine.G418 was used for selecting the positive clone.After being screened with G418,the single cell clone was sought out and cultured.The expression of IDO in HepG2 cells was detected by reverse transcription polymerase chain reaction（RT-PCR） and Western blotting.Results Not only at the level of transcription but also translation,IDO gene expressed in IDO transfectant confirmed by RT-PCR and Western blotting respectively.Conclusion Stabily transfected HepG2 of pcDNA3.1-IDO Plasmid can lead to upregulating IDO mRNA and protein expression obviously,which showes transfection HepG2 of pcDNA3.1-IDO successfully.