【摘要】 目的:构建CDK2AP1基因慢病毒过表达载体,感染人口腔鳞癌细胞系SCC-25细胞系中,为CDK2AP1基因体内外实验研究奠定实验基础。方法:将pCDH-CMV-GFP慢病毒载体Sal I和Xba I酶切位点插入CDK2AP1基因序列,构建pCDH-GFP-CDK2AP1慢病毒质粒。经PCR鉴定、测序验证CDK2AP1基因后,将其和慢病毒包装质粒混合物共同转染病毒包装细胞293TN,转染24小时后产生重组病毒GFPCDK2AP1慢病毒颗粒。经病毒浓缩纯化后,感染SCC-25口腔鳞癌细胞系并测定感染效率。结果:GFPCDK2AP1病毒中携有转染正确的CDK2AP1基因,感染人SCC-25细胞系后能稳定表达。结论:成功地在舌鳞癌细胞系SCC-25中构建了CDK2AP1基因的重组慢病毒过表达载体,为研究其在头颈鳞癌的生物学功能奠定基础。更多还原

【Abstract】 Objective:Cyclin-dependent kinase 2-associated protein 1(CDK2AP1),a cell growth inhibitory factor,is abnormally expressed in cancer cells,and might be implicated in the development of tongue squamous carcinoma cells cancer.The aim of this study is to construct the lentivirus vector containing human CDK2AP1 gene,and to examine the expression of CDK2AP1 in SCC-25 cells.Methods:The CDK2AP1 gene was cloned to SIB lentiviral expression vector by recombinant DNA technology.The positive clones were screened,and lentiviral packaged systems were co-transfected to package virus in 293TN cells by lipofectmine 2000 with target gene plasmid.Real-time PCR technique was used o test the titer of pCDH-GFP-CDK2AP1.The SCC-25 cells were transfected by pCDH-GFP-CDK2AP1 and the expression of CDK2AP1 mRNA was detected by reverse transcriptase-PCR.Results:The lentiviral vector containing CDK2AP1 was successfully constructed.The transfer efficiency of green fluorescent protein was more than 80% in the CDK2AP1 cells after transfection with pCDH-GFP-CDK2AP1 for 48 hours.Reverse transcriptase-PCR results showed that the CDK2AP1 gene expression of the transfected group was positive.Conclusion:We used the lentivirus vector construct the stable infection vector in SCC-25 cell lines.And it is useful tool to find the mechanism of CDK2AP1 in head and neck carcinogenesis.更多还原