中文摘要：

目的：构建乙型肝炎病毒X蛋白（HBx）表达载体pEYFP—Cl—X及人IGF—Ⅱ基因P4启动子调控的荧光素酶报告载体pGL3-P4，并初步探讨HBx对IGF—Ⅱ基因P4启动子活性的影响。方法：应用基因重组技术，将HBx基因及P4启动子分别克隆人pEYFP—Cl及pGL3-Basic载体，构建重组质粒pEYFP—Cl—X及pGI-3-P4；将pEYFP—Cl-X稳定转染HepG2细胞，进行G418筛选；将体外甲基化pGL3-P4瞬时转染HepG2-EYFP—X细胞，采用双荧光素酶实验检测P4启动子的转录调控活性。结果：（1）经酶切及测序鉴定，重组质粒中的目的片段HBx和P4启动子的大小分别为465bp及1246bp，序列与GenBank数据库一致。（2）获得了表达HBx的细胞系HepG2-EYFP—X，在荧光显微镜下呈现黄绿色荧光。（3）转染HepG2-EYFP—X细胞的甲基化P4启动子的荧光素酶活性明显高于其对照细胞HepG2-EYFP（P〈0．01）。结论：HBx可增加P4启动子的转录活性。

英文摘要：

AIM： To construct HBx eukaryotic expression vector pEYFP- Cl -X and eukaryotic expression vector pGL3 - P4 driven by P4 promoter of human IGF - Ⅱ gene and to investigate the effect of HBx on the transcription activity of IGF - Ⅱ gene P4 promoter. METHODS ： HBx gene and P4 promoters were cloned into pEYFP - Cl and pGL3 - basic vectors respectively by gene recombination techniques to construct recombinant plasmids pEYFP - Cl - X and pGL3 - P4. HepG2 cells were transfected with pEYFP - Cl - X and the resistant cell clones were selected by G418. Then methylated pGL3 - P4 was transiently transfected into the above cell clones, and the transcription activity of P4 promoter was determined by dual - lueiferase reporter assay system. RESULTS ： （ 1 ） Aim fragments HBx gene and P4 promoter that were cloned were 465 bp and 1 246 bp, respectively and the DNA sequences were accordant with GenBank data confirmed by restricted enzyme digestion and sequencing. （2） HepG2 - EYFP - X cells that expressed HBx protein were obtained. （ 3 ） Luciferase activity of methylated P4 promoter in the HepG2 - EYFP - X was more than that of control cell HepG2 - EYFP （P 〈0. 01 ）. CONCLUSION： HBx may enhance the transcription activity of the P4 promoter.