CAJ | 학술논문

目的: 构建乙型肝炎病毒X蛋白(HBx)表达载体pEYFP-C1-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启动子分别克隆入pEYFP-C1及pGL3-Basic载体,构建重组质粒pEYFP-C1-X及pGL3-P4;将pEYFP-C1-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465 bp及1 246 bp,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.
목적: 구건을형간염병독X단백(HBx)표체재체pEYFP-C1-X급인IGF-II기인P4계동자조공적형광소매보고재체pGL3-P4,병초보탐토HBx대IGF-II기인P4계동자활성적영향.방법:응용기인중조기술,장HBx기인급P4계동자분별극륭입pEYFP-C1급pGL3-Basic재체,구건중조질립pEYFP-C1-X급pGL3-P4;장pEYFP-C1-X은정전염HepG2세포,진행G418사선;장체외갑기화pGL3-P4순시전염HepG2-EYFP-X세포,채용쌍형광소매실험검측P4계동자적전록조공활성.결과:(1)경매절급측서감정,중조질립중적목적편단HBx화P4계동자적대소분별위465 bp급1 246 bp,서렬여GenBank수거고일치.(2)획득료표체HBx적세포계HepG2-EYFP-X,재형광현미경하정현황록색형광.(3)전염HepG2-EYFP-X세포적갑기화P4계동자적형광소매활성명현고우기대조세포HepG2-EYFP(P<0.01).결론: HBx가증가P4계동자적전록활성.
AIM: To construct HBx eukaryotic expression vector pEYFP-C1-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-C1 and pGL3-basic vectors respectively by gene recombination techniques to construct recombinant plasmids pEYFP-C1-X and pGL3-P4. HepG2 cells were transfected with pEYFP-C1-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-luciferase 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.