Construction of red fluorescent protein reporter gene vector containing HMGB1 promotor and its activity under mechanical stretch
- VernacularTitle: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
- Author:
Ning DING
;
Hui XIAO
;
Ju GAO
;
Lixin XU
;
Shouzhang SHE
- Publication Type:Journal Article
- Keywords:
high mobility group box 1 protein;
red fluorescent protein;
reporter gene;
mechanical stretch
- From:Journal of Third Military Medical University
2003;0(22):-
- CountryChina
- Language:Chinese
-
Abstract:
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,pDsRed1-1-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.
