HMGB1启动子红色荧光蛋白报告基因载体的构建及在机械牵张诱导下的活性检测

Construction of red fluorescent protein reporter gene vector containing HMGB1 promotor and its activity under mechanical stretch

目的构建高迁移率族蛋白B1(high mobility group box 1 protein,HMGB1)启动子的红色荧光蛋白报告基因载体。方法采用基因重组技术构建含HMGB1启动子区的红色荧光蛋白报告基因载体pDsRed1-1-HMGB1P。经PCR、酶切和DNA测序鉴定后,将重组载体转染HEK293细胞,在荧光显微镜下观察其在细胞内的表达以及对机械牵张刺激的反应。以转染空载体pDsRed1-1的细胞作为对照。结果PCR、酶切和DNA测序表明重组载体pDsRed1-1-HMGB1P的构建正确,该表达载体在HEK293细胞静息状态下表达水平较低,经机械牵张刺激后,在荧光显微镜下可看到高亮度的红色荧光。转染空载体pDsRed1-1的细胞未见红色荧光。结论成功构建了HMGB1启动子的红色荧光蛋白报告基因载体,对机械牵张刺激有很好的反应。

Objective To construct the red fluorescent protein reporter gene vector containing high mobility group box 1 protein（HMGB1） promoter sequence and study the regulation mechanism of the expression of HMGB1gene under mechanical stretch.Methods HMGB1 promoter was subcloned into a red fluorescent protein vector,pDsRed1-1.After identified by PCR,enzyme digestion and DNA sequencing,the recombinant vector pDsRed1-1-HMGB1P was then transfected into HEK293 cells.Blank vector or pDsRed-1 was transfected into 293 cells and served as controls. The expression of red fluorescent protein and its reaction to mechanical stretch were observed under a fluorescent microscope. HEK293 cells transfected with pDsRed1-1 vector served as control. Results PCR, double restriction enzyme digestion and DNA sequence analysis showed that the recombinant vector, pDsRedI-1-HMGB1P, was constructed correctly. This vector was lowly expressed in HEK293 cells of resting state. But after stimulated by mechanical stretch, strong red fluorescence was observed. No red fluorescence was observed in the control cells. Conclusion A red fluorescent protein reporter gene vector containing HMGB1 promoter sequence has been constructed successfully and expressed highly in mammalian cells. Since it responds to mechanical stretch effectively, it can thus provide a convenient tool to study the regulation mechanism of the expression of HMGB1 gene by mechanical stress.