人IP10启动子红色荧光蛋白报告基因载体的构建及在细胞中的表达

The Construction of Vector Containing Red Fluorescin Reporter Gene and Human IP10 Promotor and Its Expression in Cells

目的:构建IP10启动子驱动的红色荧光蛋白报告基因载体,为研究细胞内IP10的基因表达调控和相关的信号转导机制提供重要工具。方法:采用基因重组技术构建含IP10启动子区的红色荧光蛋白报告基因载体pD sRed1-1-IP10;用脂质体瞬时转染HEK293细胞,观察IP10启动子对脂多糖(LPS)刺激的反应。结果:酶切和DNA测序证明所构建的红色荧光蛋白报告基因载体是正确的;该表达载体在HEK293细胞静息状态下表达水平很低,经LPS刺激后,在荧光显微镜下可看到高亮度的红色荧光。结论:所构建的IP10启动子驱动的红色荧光蛋白报告基因载体是正确的,对生理相关刺激有很好的反应,可有效地用于IP10基因表达信号调控机制的研究。

Objective： To construct a vector containing human interferon-y-inducible protein 10 （IP10） promoter and a red fluorescent protein reporter gene, and to express it in mammalian ceils. Methods： Human IP10 promoter was subcloned into a red fluorescent protein vector, pDsRedl-1. The recombinant vector was then transfected into HEK293 cell lines. The recombinant plasmid was verified by enzyme digestion and sequence analysis, and the expression of red fluorescent protein was observed under a fluorescent microscope. Results： The expression vector was constructed correcdy, and the vector was lowly expressed in HEK293 ceils of resting state. But after the stimulation of lipopolysaccha- ride （LPS）, strong red fluorescence was observed. Conclusions： A red fluorescent protein reporter gene vector containing human IP10 promoter sequence has been constructed successfully and expressed highly in mammalian cells. Since it responds to physical stimulus effectively, it can thus provide an important and convenient tool for the study on mechanism of IP10 gene expression regulation.