截短型小鼠成纤维细胞生长因子受体-1基因慢病毒载体构建及在真核细胞中的表达  

Construction of Lentiviral Vector for Trancated Mouse Fibroblast Growth Factor Receptor-1 Gene and Its Expression in Eukaryotic Cells

作　　者：陈伟[1,2] 陈翀[1] 张焕新[1] 闫志凌[1] 程海[1] 曾令宇[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏徐州221002 [2]南京医科大学,江苏南京210029

出　　处：《中国实验血液学杂志》2012年第1期168-172,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金;编号30770915

摘　　要：本研究旨在克隆小鼠成纤维细胞生长因子受体1(fibroblast growth factor receptor 1,fgfr1)基因,构建携带增强型绿色荧光蛋白(EGFP)的截短型fgfr1(△fgfr1)重组慢病毒载体并在真核细胞中表达。采用逆转录-聚合酶链反应(RT-PCR)以BALB/c胎鼠脑组织为模板克隆全长型fgfr1基因,连接至克隆载体pCR-Blunt,通过反向PCR技术删除胞内磷酸化区域获得△fgfr1,限制性内切酶酶切后亚克隆至慢病毒转移质粒,构建携带EGFP及△fgfr1双顺反子自身失活型重组慢病毒表达质粒,通过脂质体转染法与包装质粒及包膜蛋白质粒共转染包装细胞293FT,超速离心浓缩病毒颗粒后转染293FT细胞,用荧光显微镜及流式细胞术(FCM)检测EGFP的表达,免疫印迹法(Western blot)鉴定截短型FGFR1蛋白表达。结果表明,成功克隆小鼠fgfr1基因,构建重组慢病毒转移载体LV-IRES-EGFP-△fgfr1及对照载体LV-IRES-EGFP,三质粒系统共转染293FT细胞后获得病毒滴度达到108 TU/ml。以重组病毒载体转染293FT细胞后第4天在荧光显微镜下观察到较强绿色荧光表达,FCM检测转染效率可达95%,Western blot检测显示,转染后293FT细胞表达截短型FGFR1蛋白。结论:成功构建了自身失活型慢病毒载体LV-IRES-EGFP-△fgfr1,并在真核细胞中获得表达。This study was aimed to clone the gene coding mouse flbroblast growth factor receptor-1 （fgfrl）, to construct the recombinant lentiviral vector of truncated form fgfr-1 （ afgfrl ） carrying enhanced green fluorescence protein （EGFP） and to investigate its expression in eukaryotic cells （293FT cells）. The full length fgfrl gene was cloned by RT-PCR using brain tissue of BALB/c fetal mouse as template and inserted into PCR-Blunt vector, a truncated fgfrl fragment was produced by site-directed mutagenesis for deleting intraccllular phosphorylated domain, then was subcloned into a lentiviral vector and cotransfected into 293FT packaging cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry （FCM）, and the truncated FGFR1 protein was detected by Western blot. The results demonstrated that mouse fgfrl gene was cloned and the lentiviral expression vector LV-IRES-EGFP-Afgf-rl and control vector LV-IRES-EGFP were successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10s TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in 293FT cells post transfection, and the transfection efficacy was 〉 95% determined by FCM. Expression of FGFR1 protein detected by Western blot was significantly higher than that in control group. It is concluded that the truncated gene fgfrl along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.

关 键 词：成纤维细胞生长因子受体基因 慢病毒载体构建 基因表达 293FT细胞 

分 类 号：Q7[生物学—分子生物学] R730.231[医药卫生—肿瘤]