CDK2AP1基因过表达慢病毒载体的构建与鉴定  

Construction and identification of CDK2AP1 overexpression lentivirual vector

作　　者：刘利军[1,2] 杨向明[2] 张静[2] 申致远 孙沫逸[2] 

机构地区：[1]新疆医科大学第一附属医院口腔颌面外科,新疆乌鲁木齐830011 [2]第四军医大学口腔医学院口腔颌面外科,陕西西安710032

出　　处：《现代肿瘤医学》2014年第4期751-754,共4页Journal of Modern Oncology

基　　金：国家自然科学基金项目(编号:81072230);新疆医科大学第一附属医院基金(编号:2013ZRZD14)

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。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 carci- noma 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 lenti- viral 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 tran- scriptase - 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.

关 键 词：CDK2AP1 慢病毒 过表达载体 

分 类 号：R730.23[医药卫生—肿瘤] R739.86[医药卫生—临床医学]