人细胞色素P450 2A13与GFP融合基因共表达慢病毒载体的构建与表达  

Construction and expression of CYP2A13 and GFP gene co-expressing lentiviral vector

作　　者：范晓丽[1] 谭宁[1] 孙莉[1] 

机构地区：[1]桂林医学院基础医学院组织学与胚胎学教研室,桂林541004

出　　处：《重庆医科大学学报》2011年第10期1228-1232,共5页Journal of Chongqing Medical University

基　　金：2003年广西教育厅科研基金资助项目(编号:桂教科研[2003]22号)

摘　　要：目的:构建人细胞色素P450 2A13(CYP2A13)基因与绿色荧光蛋白(Green fluorescent protein,GFP)共表达的慢病毒载体,并转染肺腺癌A549细胞使之持续高表达CYP2A13基因。方法:采用基因重组技术,将CYP2A13基因与GFP连接,构建慢病毒载体。转染293T细胞,包装后产生病毒液,测定其滴度。包装后的病毒感染A549细胞。激光共聚焦显微镜检测GFP-CYP2A13融合蛋白在A549细胞中的定位;Real-time PCR和Western blot检测A549细胞中CYP2A13表达情况。结果:成功构建人细胞色素CYP2A13基因与GFP共表达的慢病毒载体,转染293T细胞后,荧光显微镜下可见有大量的绿色荧光。激光共聚焦显微镜观察,感染后的A549细胞的胞核和胞浆中有绿色荧光,Real-time PCR和Western blot检测到A549细胞中CYP2A13的高表达。结论:成功构建了CYP2A13与GFP基因共表达的慢病毒表达载体,为研究CYP2A13基因与呼吸系统肿瘤的关系,提供了稳定的感染细胞的载体。Objective：To construct human cytochrome enzyme P450 2A13（CYP2A13） and green fluorescent protein（GFP） co-expressing lentiviral vector,and transfect A549 lung cancer cells for highly expressing CYP2A13 gene continuously.Methods：Gene recombinant technology was employed to link CYP2A13 and GFP genes for constructing lentiviral vector.After transfecting 293T cells,virus was collected and the titer wasmeasured.The packed lentiviral vector was used to infect A549 cells.Then laser scanning confocal microscope（LSCM） were applied to detect the location of fusion protein GFP-CYP2A13,while real-time PCR and Western blot were applied to deteat and the expression of CYP2A13 in the infected A549 cells respectively.Results：A recombinant lentiviral vector co-expressing CYP2A13 and GFP gene was constructed successfully.After transfection,a large number of 293T cells with green fluorescence were observed under fluorescent microscope,and green fluorescence was seen in the nucleus and cytoplasm of A549 cells under LSCM.Real-time PCR and Western blot showed that CYP2A13 gene was highly expressed.Conclusion：Successful construction of human CYP2A13 and GFP co-expressing lentiviral vector provides a stable vector for further investigation of the relationship between CYP2A13 gene and respiratory tumors.

关 键 词：CYP2A13 绿色荧光蛋白 慢病毒载体 转染 

分 类 号：Q782[生物学—分子生物学]