中文摘要：

目的构建p55PIK高表达的质粒,筛选稳定高表达p55PIK的LoVo细胞系,检测其对LoVo细胞周期的影响。方法以基因组cDNA为模板,聚合酶链反应（PCR）扩增目的片段,通过限制性核酸内切酶进行酶切,T4DNA连接酶将目的片段插入pcDNA3.1质粒中;将携带p55PIK基因的pcDNA3.1质粒转染LoVo细胞,加入适量的G418筛选（500μg/mL）,并通过实时定量PCR（qPCR）和Western blot方法检测p55PIK mRNA水平和蛋白水平,进一步通过BrdU/PI掺入法检测p55PIK对LoVo细胞周期和DNA合成的影响。结果成功构建了p55PIK高表达的质粒,并筛选出稳定高表达p55PIK的LoVo细胞系;在LoVo细胞中高表达p55PIK能够促进细胞S期的进程和DNA的合成。结论成功构建了pcDNA3.1-p55PIK质粒,并筛选出p55PIK稳定高表达的LoVo细胞系,高表达p55PIK能够促进LoVo细胞S期的进程和DNA的合成。

英文摘要：

Objective To construct the p55PIK high-expression plasmid,screen out LoVo cell line with stable high-expres- sion of p55PIK,and examine the effect of p55PIK on cell cycle of LoVo cells. Methods Target fragment was amplified by polymerase chain reaction（PCR）,with the genomic eDNA as the template,and then cloned into the pcDNA3.1 vector by using restriction endonuclease and T4 DNA ligase. LoVo cells were transfected with pcDNA3.1-p55PIK vector and then G418（500 μg/mL）was added to identify stable overexpressed p55PIK cell line. Real-time quantative PCR and Western blot were used to detect the mRNA and expression levels of p55PIK. Furthermore,the effect of p55PIK on cell cycle progression and DNA synthesis was examined by BrdU/PI incorporation method. Results p55PIK high-expression vector was successfully constructed and LoVo cells with stable high-expression of p55PIK screened out. Over-expressed p55PIK in LoVo cells could promote cell cycle progression in S phase and DNA incorporation in LoVo cells. Conclusion Stable LoVo cell line with p55PIK high expres- sion was successfully obtained. Overexpressing p55PIK could promote cell cycle progress in S phase and DNA incorporation in the cells.