人I型干扰素受体亚基稳定表达真核细胞的筛选及鉴定

Construction of pcDNA3.1-IFNAR1 Eukaryotic Expression Plasmid and its Expression in HepG2 Cell Lines

目的:筛选及鉴定人I型干扰素受体亚基(IFNAR1)稳定表达真核细胞。方法:体外扩增IFNAR1基因片段,将双酶切后扩增片段和pc DNA3.1(+)连接构建重组质粒pc DNA3.1-IFNAR1,转化BL21感受态细菌培养,提取重组质粒行琼脂糖凝胶电泳和测序鉴定。重组质粒pc DNA3.1-IFNAR1转染肝癌细胞系Hep G2,新霉素筛选挑取阳性克隆细胞,蛋白质印迹试验(Western blotting)分析阳性细胞IFNAR1表达水平。结果:经双酶切和测序验证pc DNA3.1-IFNAR1重组质粒构建成功。pc DNA3.1-IFNAR1转染Hep G2经新霉素筛选后获得稳定表达细胞系,蛋白质印迹试验(Western blotting)证实这些细胞具有较好蛋白表达水平。结论:体外成功构建不同IFNAR1稳定表达水平的Hep G2细胞。

Objective To identify the expression of IFNAR1 in Human hepG2 cells and detected its expression. MethodsThe full-length IFNAR1 cDNA gene in HepG2 cell lines was amplified by RT-PCR and then integrated into pcDNA3.1（＋） to construct the expression vector pc DNA3.1-IFNAR1. Subsequently,the pc DNA3.1-IFNAR1 was transfected into HepG2 cell and selected with neomycin. The positive cell clones were picked up and the expression of IFNAR1 protein was detected by western blotting method. Results pc DNA3.1-IFNAR1 was successfully constructed and demonstrated by agrose electrophoresis and sequencing. The different expression of IFNAR1 protein was verifi ed in stable HepG2 cells. Conclusion IFNAR1 protein can stably be expressed in HepG2 cells transfected with pc DNA3.1- IFNAR1 vector.