pHSP70P-EGFP真核表达载体的构建及其在人Chang's肝细胞中的表达

Construction of eukaryotic expression vector pHSP70P-EGFP and its expression in human Chang′s liver cell

目的构建HSP70启动子与绿色荧光蛋白重组真核表达载体,并在人Chang's肝细胞表达,建立一种新的体外药物肝毒性早期预测细胞模型。方法提取人Chang's肝细胞基因组DNA,钓取HSP70启动子基因,构建重组载体,经脂质体转染人Chang's肝细胞,用G418筛选稳定转染细胞,用荧光显微镜观察绿色荧光表达情况,用实时荧光定量PCR检测EGFP基因表达量的变化。结果成功钓取HSP70启动子基因并导入真核表达载体pEGFP-N1;G418以300 ng/mL的终浓度筛选出稳定转染的单克隆细胞;荧光显微镜观察到肝毒性药物酮康唑刺激人Chang's肝细胞后,可诱导HSP70启动子调控增强型绿色荧光蛋白发出绿色荧光,药物刺激组EGFP mRNA相对表达量与正常对照组比较有统计学意义(t=-14.21,P<0.05)。结论成功构建HSP70启动子与绿色荧光蛋白共表达真核表达载体,获得稳定转染单克隆细胞,并证实HSP70在肝毒性药物刺激下的应激表达,为建立新的体外药物肝毒性早期预测细胞模型进行高通量筛选新药奠定了基础。

Objective To construct the eukaryotic expression vector of HSP70 promoter and green fluorescent protein and expression the recombine proteins in human Chang′s liver cells in order to establish a new drugs hepatotoxicity cell model in vitro for early prediction.Method The genome DNA of human Chang′s liver cell was extracted and the promoter of HSP70 gene was obtained.HSP70 promoter gene was transfected to human Chang′s liver cell by means of pEGFP-N1 vector with liposome.The steadily transfected cells were screened by G418 selection.The expression of green fluorescent protein was observed by fluorescence microscope.The change of the expression of EGFP gene were observed by real-time fluorescence quantitative PCR.Results pEGFP-N1 vector expressing recombinant HSP70 promoter and green fluorescent protein was successfully constructed and the steadily transfected monoclonal cells were screened by G418 in final concentration of 300 ng/mL.The stimulation of hepatotoxic drug Ketoconazole to human Chang′s liver cell induced enhancement of green fluorescence regulated by HSP70 promoter.The relative expression of EGFP mRNA by stimulation of drug in the transfected cells was significantly higher than that of the control group（t=-14.21,P0.05）.Conclusion The eukaryotic expression vector of HSP70 promoter and green fluorescent protein was successfully constructed and the monoclonal cells with stable transfection were obtained.The stress expression of HSP70 by stimulation of hepatotoxic drugs was verified.A cell mode for early prediction of hepatotoxic drugs was provided to select new drugs with high-throughput in vitro.