Med19过表达慢病毒载体的构建及鉴定  

Construction and identification of a recombinant lentiviral expression vector carrying on Med19 gene and its expression in 293T cells

作　　者：王晓艳[1] 李莉华[1] 郭子健[1] 饶敏[1] 

机构地区：[1]苏州大学附属第四医院肿瘤研究所,江苏省无锡市214062

出　　处：《实用医学杂志》2011年第22期4013-4015,共3页The Journal of Practical Medicine

基　　金：无锡市卫生局项目(编号:XM1012)

摘　　要：目的：构建含Medl9基因的过表达慢病毒载体。方法：用PCR技术获得Medl9基因片段，将其连接入酶切后的线性慢病毒载体．转化感受态细胞进行PCR及测序鉴定。脂质体转染法共转染293T细胞，荧光显微镜及Westernblot检测转染效率。包装成慢病毒，实时定量PCR检测病毒滴度。结果：成功获取Medl9基因。测序证实所获取基因序列完全正确。荧光显微镜以及WesternNot检测均证实pGC-FU-Med19携有正确的Medl9基因，并能在293T细胞中表达。实时定量PCR检测病毒滴度为2×10^9TU／mL。结论：成功构建Medl9基因慢病毒表达载体，为进一步研究其生物学功能奠定了基础。Objective To construct a lentiviral expression vector of human Medl9 and identify its expression in 293T cells. Methods ttuman Med19 sequence was amplified, purified, ligated with lentiviral vector plasmid and verified by sequencing. The verified plasmids were transfected into 293T cells by Lipofectamine 2000. Transfection efficiency was assayed by both immunofluorescence microscopy and western blot. Medl9 lentiviral vector plasmid from the selected constructs was propagated and harvested with a virus packaging system, and the virus titers were determined. Results The Med19 gene was successfully constructed into pGC-FU-Medl9 express lentiviral vector. Medl9 expression was observed using fluorescence microscope after the transfection. Western blotting also showed Med19 expression in the transfected 293T cells. The Medl9 gene sequence of the vector was successfully verified by sequencing. The concentrated titer of virus suspension was 2×10^9 Tu/mL. Conclusion The human Medl9 lentiviral expression vector wa,~ successfully constructed. This facilitates further studying Medl9 biological function.

关 键 词：MED19 慢病毒 过表达 

分 类 号：R730.2[医药卫生—肿瘤]