FGF4基因慢病毒表达载体的构建、包装及鉴定  

Construction,packaging and identification of a lentiviral vector carrying the fibroblast growth factor 4 gene

作　　者：徐丽娟[1] 张云巍 胡亚卓[2] 阎丽[1] 

机构地区：[1]中国人民解放军总医院南楼临床部消化内科,北京市100853 [2]中国人民解放军总医院老年医学研究所病理科,北京市100853

出　　处：《世界华人消化杂志》2015年第17期2768-2773,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30900669;北京市科技新星计划基金资助项目;No.2011117~~

摘　　要：目的:构建成纤维细胞生长因子4(fibroblast growth factor 4,FGF4)基因慢病毒过表达载体并对其进行鉴定、包装.方法:采用Bam HⅠ/AgeⅠ酶酶切含目的基因FGF4的质粒,将目的基因与酶切线性化的载体进行定向交换,构建重组慢病毒表达载体p GC-FU-FGF4,将其产物转化大肠杆菌感受态细胞.筛选阳性克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序分析,将目的质粒感染293T细胞24 h后,采用Realtime定量PCR测定包装的病毒滴度.结果:PCR结果显示扩增的目的基因FGF4已成功插入p GC-FU载体.阳性克隆测序结果显示与目的基因序列一致.目的质粒转染后24 h,293T细胞几乎100%表达绿色荧光,Real-time定量PCR法测定包装的病毒滴度为2×108 TU/m L.结论:本实验成功构建了FGF4慢病毒表达载体,成功对慢病毒及进行了包装及病毒滴度测定.AIM：To construct,package and identify alentiviral vector carrying the fibroblast growth factor 4 gene（p GC-FU-FGF4）.METHODS：A plasmid containing the FGF4 gene was digested using BamH Ⅰ/Age Ⅰ restriction enzymes,and the target gene fragment was cloned into the p GC-FU vector to result in a recombinant lentiviral vector（p GC-FU-FGF4）.p GC-FU-FGF4 was then transformed into competent Escherichia coli cells,and the positive clones were identified by PCR.The identified recombinant vector was used to infect 293 T cells.The titer of packaged virus was determined using real-time quantitative PCR 24 h after infection.RESULTS：PCR analysis showed that the amplified target gene was inserted in the p GCFU vector.Digestion analysis showed that the reconstructed plasmid was consistent with the theoretical fragment,and the sequencing result showed that the positive fragment was exactly the same as the target gene.Twenty-four hours after infection,almost 100% of cells displayed green fluorescence.Real-time quantitative PCR assay showed that the packaged viral titer was 2×108 TU/m L.CONCLUSION：A lentiviral vector carrying the FGF4 gene has been successfully constructed.

关 键 词：慢病毒载体 成纤维细胞生长因子4基因 感受态细胞 

分 类 号：R575.2[医药卫生—消化系统]