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背景与目的:在前期工作中,我们经计算机序列分析发现,在乙型肝炎病毒(hepatitisBvirus,HBV)基因组增强子Ⅰ上游1047～1059bp存在P53样应答元件结合序列5’-TGCC(G)TTGCCT-3’,体外实验应用凝胶迁移方法(EMSA和EMSSA)发现这段序列与P53蛋白能够进行特异性结合。本实验拟观察肝癌细胞中P53蛋白与HBV基因组P53样应答元件结合序列之间的关系。方法:报告基因质粒pX-CAT上游为X基因增强子和启动子序列,其中含有P53样应答元件结合序列5’-TGCGTTGCCT-3’。首先用PCR方法将pX-CAT质粒中5’-TGCGTTGCCT-3’进行点突变,成为5’-TGTATTGTAT-3’,以破坏P53样应答元件结合序列。将pX-CAT和变异后的pX-CAT(mpX-CAT)分别单独或与pCMV-p53质粒共转染HepG2细胞,通过检测报告基因CAT的活化情况,观察P53蛋白与这段DNA序列的作用关系。将HBV基因P53样应答元件结合序列反向构建于真核表达质粒pZeoSV2中(αpZeoXP),转染HepG2.2.15细胞并稳定筛选,以封闭HBV基因上P53样结合位点,观察细胞中P53/P21蛋白表达的改变,以及细胞周期、细胞凋亡的变化。结果:HepG2细胞中,报告基因CAT活性在pCMV-p53与pX-CAT共转染组较pX-CAT单独转染组明显升高(1.353vs0.738,P<0.05),而mpX-CAT单独转染组CAT活性则明显降低(0.304),即使与pCMV-p53共转染CAT活性?
BACKGROUND & OBJECTIVE: In the previous work, we found that P53-like response element binding sequence 5’-TGCC (G) TTGCCT-1 was located upstream of 1047-1059bp of hepatitis B virus genomic enhancer Ⅰ, 3 ’, in vitro experiments using gel migration method (EMSA and EMSSA) found that this sequence and P53 protein can be specifically bound. The experiment to observe the relationship between P53 protein and HBV genome P53-like response element binding sequence in hepatocellular carcinoma cells. Methods: The upstream of the reporter gene plasmid pX-CAT was the X-enhancer and promoter sequence containing the P53-like response element binding sequence 5’-TGCGTTGCCT-3 ’. The 5’-TGCGTTGCCT-3 ’in the pX-CAT plasmid was first point-mutated by PCR into 5’-TGTATTGTAT-3’ to disrupt the P53-like response element binding sequence. The pX-CAT and mutated pX-CAT (mpX-CAT) were cotransfected into HepG2 cells alone or with the pCMV-p53 plasmid respectively. The effect of the P53 protein on this DNA sequence was examined by detecting the activation of the reporter gene CAT . The P53-like response element binding sequence of HBV gene was reversely constructed in the eukaryotic expression plasmid pZeoSV2 (αpZeoXP), transfected into HepG2.2.15 cells and stably screened to block the P53-like binding site on the HBV gene. The expression of P53 / P21 Protein expression changes, and cell cycle, apoptosis changes. Results: The CAT activity in HepG2 cells was significantly higher than that in pX-CAT transfected group (1.353vs0.738, P <0.05) CAT activity was significantly decreased (0.304) in the dyed group, even though CAT activity was co-transfected with pCMV-p53?