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目的探讨人肝再生增强因子(ALR)对钙结合蛋白S100A11启动子(S100A11p)转录的调节作用。方法利用生物信息学技术确定S100A11p区域,聚合酶链反应(PCR)扩增S100A11p,克隆至真核报告载体pCAT3中,构建pCAT3S100A11p报告载体;以该质粒转染HepG2细胞系,用酶联免疫吸附法(ELISA)检测报告基因编码产物氯霉素乙酰转移酶(CAT)的表达活性,并以人ALR的真核表达载体pcDNA3.1(-)ALR共转染的HepG2细胞系,用酶联免疫吸附法(ELISA)检测CAT的表达活性。结果pCAT3S100A11p在HepG2细胞中具有CAT的表达活性;以pcDNA3.1(-)ALR和pCAT3S100A11p共转染HepG2细胞,CAT表达活性是单独转染细胞组pCAT3S100A11p的0.42倍。结论所克隆的S100A11启动子有启动子的转录活性,ALR的转基冈表达具有对S100A11基因的下调作用。
Objective To investigate the regulatory effect of human augmenter of liver regeneration (ALR) on S100A11 promoter (S100A11p) transcription. Methods The S100A11p region was identified by bioinformatics technique. S100A11p was amplified by polymerase chain reaction (PCR) and cloned into eukaryotic reporter vector pCAT3 to construct pCAT3S100A11p reporter vector. The plasmid was transfected into HepG2 cell line and detected by enzyme-linked immunosorbent assay (ELISA) was used to detect the expression activity of the gene encoding the product chloramphenicol acetyltransferase (CAT) and co-transfected with the human ALR eukaryotic expression vector pcDNA3.1 (-) ALR HepG2 cell line, with enzyme-linked immunosorbent assay The activity of CAT was detected by ELISA. Results pCAT3S100A11p had the activity of CAT expression in HepG2 cells. The co-transfection of HepG2 cells with pcDNA3.1 (-) ALR and pCAT3S100A11p resulted in a 0.42-fold higher CAT activity than that of pCAT3S100A11p transfected alone. Conclusion The cloned S100A11 promoter has the transcriptional activity of the promoter, and the knockdown of ALO has the effect of down-regulating the S100A11 gene.