Pokemon表达慢病毒载体的构建及鉴定.pdfVIP

332 Medical Science Journal of Central South China,July2013,Vol.41,No.4 文章编号:2095-1116(2013)04-0332-04 ·基础医学· Pokemon表达慢病毒载体的构建及鉴定 祖旭宇,刘摇 文,谭晶晶 (南华大学附属第一医院临床医学研究所,湖南衡阳421001) 摘摇 要:摇 目的摇 构建Pokemon表达慢病毒载体。 摇 方法摇 通过PCR 扩增Pokemon cDNA,将Pokemon cDNA 连接于GV165载体,经测序确认后,将GV165/ Pokemon与pHelper 1.0和pHelper2.0共转染至293T 细胞中,收获 病毒,通过Real time PCR测定滴度;将Pokemon表达慢病毒载体侵染293T 细胞通过免疫荧光和Western blot检测 Pokemon表达慢病毒载体的转染效率和Pokemon 的表达能力。 摇 结果摇 在感染Pokemon 慢病毒载体293T 细胞中 能检查到Pokemon鄄GFP 融合蛋白的表达。 摇 结论摇 成功构建表达Pokemon 的慢病毒载体。 关键词:摇 Pokemon;摇 慢病毒载体;摇 基因治疗;摇 293T 细胞 中图分类号:R737.9摇 摇 摇 文献标识码:A Establishment and Identification of Lentivirus with Pokemon Expression ZU Xuyu,LIU Wen,TANJingjing (Institute of ClinicalMedicine,the FirstAffiliated Hospital,University of South China, Hengyang,Hunan421001,China) Abstract:摇 Objective摇 To obtain Lentivirus with Pokemon expression.摇 Methods摇 Pokemon cDNA was obtained from cDNA pool by RT鄄PCR,and the Pokemon cDNA was ligatedwith GV165 vector;the GV165/ Pokemon plasmid con鄄 firmed by sequencingwascotranfectedwithpHelper 1.0 andpHelper2.0 into293T cellstoobtainPokemonLentivirus;ti鄄 terof LentiviruswithPokemonexpressionwasassessedby RealTimePCR;theinfectionefficiencyofPokemonLentivirusin 239T cellswas monitored by using immuno鄄microscope and the expression of Pokemon was detected by Western blot in Pokemon Lentivirus infected239T cells.摇 Results摇 Pokemon Lentivirus could expressPokemon in239T cells.摇 Conclu鄄 sion摇 Lentiviruswith the expression of Pokemon was successfully established. Key words:摇 Pokemon;摇 Lentivirus;摇 Gene therapy;摇 293T cell [1] [2] [3]