表达3个外源基因真核表达载体的构建

Construction of a eukaryotic expression vector expressing three exogenous genes

为构建可以同时表达3个外源基因的真核表达载体,本实验以pCAGGs载体为骨架,引入SV40启动子及单疱疹病毒(HSV)TK基因的polyA序列,并且在β-actin启动子下游插入内部核糖体进入位点序列(IRES),构建了能够同时表达3个外源基因的真核表达质粒pCAGGs-IRES。本研究将PR8流感病毒的M基因、绿色荧光蛋白基因EGFP及H1N1猪流感病毒的HA基因,分别克隆于pCAGGs-IRES载体的3个启动子下游,转染细胞后,通过间接免疫荧光和western blot表明本实验构建的pCAGGs-IRES载体能同时表达3个外源基因。该载体有望为基因疫苗、基因操作及蛋白质相互作用等研究提供新的真核表达载体。

To construct the eukaryotic expression vector for three exogenous genes expression, the pCAGGs-IRES was construct based on pCAGGs by inserting a SV40 promoter and the polyA sequence of TK gene from herpes simplex virus fragment and introducing a internal ribosome entry site （IRES） sequence under β-actin promoter. To verify the feasibility of the new vector, the M gene of influenza PR8 virus, EGFP gene and HA gene of H1N1 influenza virus were inserted into pCAGGs-IRES under the promoters of SV40, β-actin, and IRES. The recombinant plasmid was transfected into MDCK or 293T cells, and the exogenous genes were successfully expressed by detecting with indirect immunofluorescence assay and western blot. This improved plasmid would be a useful eukaryotic expression vector for DNA vaccine and other studies.