乙型肝炎病毒X蛋白上调S100钙结合蛋白A11基因启动子表达活性研究

The up-regulating effect of hepatitis B virus X protein on calgizzarin S100A11 gene promoter expression activity

目的 探讨乙型肝炎病毒 (HBV)X蛋白 (HBV X蛋白 )对S10 0钙结合蛋白A11(calgizzarinS10 0A11)启动子转录的激活作用。方法 以解放军第 30 2医院基因治疗研究中心自行构建的HBV X蛋白反式调节基因的cDNA文库抑制性消减杂交 (SSH)技术的筛选结果及基因表达谱芯片结果为基础 ,利用生物信息学技术确定S10 0A11的启动子区域 (S10 0A11 p) ,聚合酶链反应 (PCR)扩增S10 0A11 p,克隆至真核报告载体pCAT3中 ,构建pCAT3 S10 0 p报告载体 ;以该质粒转染肝癌细胞系HepG2细胞系 ,用酶联免疫吸附法 (ELISA)检测氯霉素乙酰转移酶 (CAT)的表达活性 ;并与pcDNA3.1( ) X共转染HepG2细胞系 ,用ELISA法检测CAT的表达活性。结果 pCAT3 S10 0 p在HepG2细胞中能够指导CAT的表达 ;共转染实验中pCAT3 S10 0 p+pcDNA3.1( ) X组CAT的表达活性是pCAT3 S10 0 p的 2 .1倍。结论 所克隆的S10 0A11启动子有顺式激活下游基因的活性作用 ;HBV的X蛋白具有对S10 0A11的反式激活作用。本实验进一步验证了解放军第 30 2医院基因治疗研究中心利用SSH技术及基因表达谱技术研究HBV

Objective To investigate the activating effect of HBV X protein on transcription of calgizzarin S100A11 gene promoter. Methods The sequence of calgizzarin S100A11 gene promoter was identified in GenBank by bioinformatics and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was cloned into pCAT3 reporter vector. The HepG2 cells were transfected by pCAT3-S100-p, and then co-tranfected by pCAT3-S100-p and pcDNA3.1(-)-X. The choloraphenical acetyltransferase(CAT) activity was detected by enzyme-linked immunosorbent assay(ELISA) kit. Results It was shown that pCAT3-S100-p could regulate the expression of CAT in HepG2 cells. The expression of CAT in co-transfection of pCAT3-S100-p and pcDNA3.1(-)-X was 2.1 fold higher than pCAT3-S100-p plasmid. Conclusion HBV-X protein can trans-activate the expression of S100A11 protein, and it further proved our previous results by SSH and biochips.