盘状结构域受体1过表达慢病毒载体的构建及稳定过表达人胃腺癌BGC823细胞系的建立

Construction of a lentiviral vector over-expressing DDR1 and establishment of a BGC823 cell line stably over-expressing DDR1

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 m RNA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 se-quencing 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.