目的 构建盘状结构域受体1 (DDR1)基因的慢病毒载体,建立稳定过表达DDR1的人胃腺癌BGC823细胞系.方法 实时荧光定量PCR扩增DDR1目的基因,双酶切目的基因并插入LV5载体,构建LV5-DDR1慢病毒表达载体;随后转入人胚肾细胞293FT中进行慢病毒包装,用获得的慢病毒毒液感染人胃腺癌BGC823细胞系并通过嘌呤霉素筛选阳性表达细胞;根据转染情况,将细胞分为BGC823组(空白BGC823细胞)、BGC-NC组(转染空载慢病毒LV5的BGC823细胞)、BGC-DDR1组(转染过表达LV5-DDR1的BGC823细胞),分别用实时荧光定量PCR和蛋白免疫印迹法分析DDR1 mRNA和蛋白的表达.结果 经限制性内切酶鉴定及测序分析,成功构建了LV5-DDR1慢病毒表达载体并进行慢病毒包装.实时荧光定量PCR和蛋白免疫印迹法检测结果显示,与BGC-NC组比较,转染LV5-DDR1慢病毒表达载体组的BGC823细胞DDR1表达水平明显升高(P＜0.01).结论 成功构建了DDR1慢病毒表达载体及DDR1稳定过表达的BGC823细胞系.%Objective To construct a lentiviral vector over-expressing DDR1 and establish a BGC823 cell line stably over-expressing DDR1.Methods The target DDR1 gene was amplified by real-time polymerase chain reaction (RT-PCR),and then was digested and inserted into a LV5 plasmid to construct an LV5-DDR1 lentiviral vector.After the lentiviral vector was packaged,BGC823 cell line infected by the packaged virus was selected by puromycin.Cells were divided into three groups,i.e.BGC823 (BGC823 cells without transfection),BGC-NC (BGC823 cells transfected by LV5) and BGC-DDR1 (BGC823 cells transfected by LV5-DDR1),and the expression of DDR1 in each group was examined by RT-PCR and Western blot.Results DNA sequencing confirmed that the lentiviral vector overexpressing DDR1 was successfully constructed.RT-PCR and Western blot analyses showed that the expression of DDR1 was significantly higher (P ＜ 0.01) in the BGC823 cell line transfected by LV5-DDR1 than in the BGC-NC group.Conclusion A lentiviral vector overexpressing DDR1 and BGC823 cell line stably over-expressing DDR1 were successfully constructed.
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