Med19过表达慢病毒载体的构建及鉴定

目的:构建含Med19基因的过表达慢病毒栽体。方法:用PCR技术获得Med19基因片段,将其连接入酶切后的线性慢病毒栽体,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Western blot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Med19基因,测序证实所获取基因序列完全正确。荧光显微镜以及Western blot检测均证实pGC-FU-Med19携有正确的Med19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Med19基因慢病毒表达栽体,为进一步研究其生物学功能奠定了基础。

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

Objective To construct a lentiviral expression vector of human Med19 and identify its expression in 293T cells.Methods Human 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 s...