【摘要】 目的:通过构建S100P慢病毒表达载体,研究其对结肠癌Caco-2细胞的影响。方法:以DLD-1细胞cDNA为模版,PCR扩增S100P基因序列,然后将该序列克隆插入慢病毒载体中,构建S100P慢病毒表达载体,包装产生慢病毒颗粒。将慢病毒颗粒感染Caco-2细胞,荧光显微镜观察细胞绿色荧光蛋白表达;应用逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Western blot)技术检测S100P的表达情况;MTT法检测细胞生长变化;细胞平板克隆培养检测其细胞克隆形成能力。结果:构建的慢病毒表达载体Lenti-S100P经酶切和测序鉴定结果正确;携带S100P基因的慢病毒颗粒感染Caco-2后,细胞中S100P基因和蛋白过表达,促进细胞克隆形成能力。结论:本研究成功构建S100P慢病毒表达载体,为深入研究S100P基因的相关功能提供了一定的实验基础。更多还原

【Abstract】 Objective: To construct a lentiviral vector containing S100P gene and study its effect on colon cancer Caco-2 cells.Methods: S100P was amplified from cDNA of DLD-1,and inserted into lentiviral expression vector to generate Lenti-S100P vector.The inserted S100P gene was verified by double enzyme digestion and DNA sequencing.Subsequently,lentiviruses were produced and transduced into Caco-2 cells.EGFP expression was examined under fluorescence microscopy,the expression of S100P was detected with semi-RT-PCR and Western blot.Cell growth and colony forming ability were detected with MTT and plate cloning technique.Results: The lentiviral S100P expression vector was successful constructed.Overexpression of S100P was verified in Caco-2 cells with Lenti-S100P.S100P promoted Caco-2 cells colony forming ability,while no significant effect on cell growth.Conclusion: Lenti-S100P vector is successfully constructed,and it will provide a basis for further study of S100P gene function.更多还原