Peroxiredoxin 2基因慢病毒载体的构建及对结直肠癌SW480细胞增殖的影响

摘要

To construct the lentiviral vector containing Peroxiredoxin 2(Prdx2) gene and the colorectal cancer cell line stably transduced with Prdx 2-containing vector , so as to provide a useful tool for studying the role of Prdx 2 in colorectal cancer.Methods: Prdx2 was amplified by PCR and inserted into lentiviral expression vector Ubi-MCS-EGFP-IRES-Puromycin (GV218) to generate Ubi-Prdx2-EGFP-Puromycin(LV-Prdx2) vector.The inserted Prdx2 gene was verified by double enzyme digestion and DNA sequencing.Subsequently ,lentiviruses were produced and transduced into SW 480 cells.EGFP expression was examined under fluorescence microscopy ,the expression of Prdx2 was detected with qRT-PCR and Western blot.Cell growth and colony forming ability were detected with MTT and plate cloning technique.Results: The lentiviral Prdx2 expression vector was successful construc-ted.Overexpression of Prdx2 was verified in SW480 cells with LV-Prdx2 vector.Prdx2 promoted SW480 cell growth and colony forming ability(P<0.05).Conclusion:Ubi-Prdx2-EGFP-Puromycin(LV-Prdx2) vector is successfully constructed,and the SW480/LV-Prdx2 cell line with stable transduction of Prdx2 containing vector is established.Overexpression Prdx2 can significantly promote the proliferation of colorectal cancer SW 480 cells.%目的：构建Peroxiredoxin 2（Prdx2）基因慢病毒表达载体，建立能够稳定高表达Prdx2的结直肠癌SW480细胞株，研究Prdx2的高表达对SW480细胞生长增殖的影响。方法：利用PCR扩增Prdx2编码区片段，克隆插入载体Ubi-MCS-EGFP-IRES-Puromycin （GV218）中，构建Ubi-Prdx2-EGFP-Puromycin（LV-Prdx2）慢病毒表达载体，包装产生慢病毒颗粒，鉴定后将其转染SW480细胞，荧光显微镜观察细胞绿色荧光蛋白表达；应用实时荧光定量逆转录聚合酶链反应（ qRT-PCR ）和Western blot方法检测SW480细胞中Prdx2的mRNA和蛋白水平表达情况；MTT法检测细胞生长情况；平板克隆形成实验检测细胞克隆形成能力。结果：构建的重组慢病毒表达载体Ubi-Prdx2-EGFP-Puromycin（LV-Prdx2）经鉴定正确；慢病毒转染SW480细胞后，细胞中Prdx2在mRNA和蛋白水平高表达。 MTT法及平板克隆形成实验结果显示Prdx2高表达的SW480细胞增殖能力显著增强（P＜0.05）。结论：成功构建Prdx2基因慢病毒表达载体Ubi-Prdx2-EGFP-Puromycin，筛选出稳定高表达Prdx2的SW480细胞株；高表达的Prdx2分子可显著促进结直肠癌SW480细胞增殖。