高迁移率族蛋白B1真核表达载体的构建及其对肿瘤坏死因子-α报告基因活性的影响

Construction of high mobility group box-1 protein eukaryotic expression vector and its effect on tumor necrosis factor-α reporter gene activity

目的克隆人的高迁移率族蛋白B1(HMGB1)基因,插入pc-DNA3载体中并检测其对肿瘤坏死因子-α(TNF-α)报告基因活性的影响。方法采用聚合酶链反应(PCR)技术,从人乳腺癌细胞系MCF-7基因组中扩增出HMGB1基因,插入载体pc-DNA3中,确定所扩增的DNA序列;用蛋白质免疫印迹法(Western blotting)在293T细胞中检测其表达;应用报告基因方法检测其对TNF-α报告基因表达的影响。结果酶切和测序结果表明扩增的HMGB1序列正确,大小为648bp。在293T细胞中正确表达相对分子质量约24000的HMGB1蛋白。下游基因TNF-α荧光素酶活性实验显示,构建的真核表达载体在内毒素刺激后以先增加后降低的方式影响TNF-α的活性,且对从95-120长度的TNF-α报告基因活性影响最明显。结论HMGB1以先增加后降低的方式影响TNF-α基因的表达,且主要通过26个碱基大小的片段发挥作用。

Objective To clone human high mobility group box-1 protein （HMGB1） gene and investigate its effect on tumor necrosis factor-α （TNF-a） reporter gene activity. Methods The human HMGB1 gene was amplified from human breast cancer cell line MCF-7 genomic DNA by polymerase chain reaction （PCR） and cloned into pc-DNA3. The resulting plasmid was determined by DNA sequencing. HMGB1 protein expression was identified by Western bloting in 293T cell line. Reporter gene detection was applied to analyze the influence of HMGB1 on TNF-α gene expression. Results The results of DNA sequencing and enzyme digestion showed that the cloned HMGB1 gene was correct （648 bp）. HMGB1 protein was expressed in 293T cell to be approximately 24 000. TNF-α reporter gene activity was increased, peaking at 300 μg/L HMGB1, and then decreased at a dose exceeding 500 μg/L HMGB1. 95-120 bp TNF-α reporter gene activity was the highest. Conclusion HMGB1 can markedly regulate TNF-α gene expression in an up-to-down manner, which might be significantly affected by fragment 95-120 bp in TNF-α promoter.