乙型肝炎病毒X蛋白对B7-H6基因的激活作用

摘要

目的 探讨乙型肝炎病毒X蛋白(HBx)对人B7-H6基因的激活作用.方法 提取人基因组DNA作为模版,聚合酶链反应(polymerase chain reaction,PCR)获取约2.2 Kb B7-H6基因启动子DNA片段,扩增产物经Kpn Ⅰ和Hind Ⅲ双酶切后插入到pGL3-Basic中构建双荧光素酶报告基因pGL3-B7-H6,测序正确后分别与HBV病毒蛋白S、C和X的真核表达质粒pCMV-HBs、pCMV-HBc和pCMV-HBx共转染HepG2细胞,检测荧光素酶活性变化,然后用不同剂量的pCMV-HBx表达质粒与pGL3-B7-H6共转染HepG2细胞,Western blot检测B7-H6蛋白表达水平.结果 本研究克隆出的B7-H6基因启动子片段序列与GenBank中记录一致,pGL3-B7-H6构建成功.pGL3-B7-H6启动子质粒转染HepG2细胞荧光素酶活性较转染空载体pGL3-Basic组显著增加(5.24 ±1.25 vs.1.12 ±0.31,P=0.005).pGL3-B7-H6启动子质粒与pCMV-HBx质粒共转染HepG2细胞,荧光素酶活性较对照组显著增加(17.60±3.36 vs.6.73±1.36,P=0.001).Western blot结果显示,HBx蛋白可显著增强B7-H6蛋白的表达.结论 本研究发现HBx蛋白可能增强B7-H6基因启动子的转录活性并促进B7-H6蛋白的表达.%Objective To investigate the key factor(s) of hepatitis B virus X protein (HBx) promoting B7-H6 gene activation.Methods The DNA fragments of the B7-H6 promoter were amplified from the human genomic DNA using polymerase chain reaction (PCR).Products of PCR were digested by KpnI and Hind Ⅲ,and inserted into luciferase reporter vector (pGL3-Basic).The correctness of the recombinant plasmid pGL3-B7-H6 was confirmed by sequencing.Human hepatoma cell line HepG2 were cotransfected with pGL3-B7-H6 and the eukaryotic expression vectors (pCMV-HBs、pCMV-HBc and pCMV-HBx),and the relative luciferase activity was detected.The different dose of HBx expression plasmids were transfected into the HepG2 cells,and the expression of B7-H6 protein were determined by Western blot.Results A period of 2.2 Kb of B7-H6 promoter region were successfully cloned into pGL3-Basic,which was confirmed by DNA sequencing.The relative luciferase activity was significantly higher in the cells transfected with pGL3-B7-H6 than that in the cells transfected with the empty vector pGL3-Basic (5.24 ± 1.25 vs.1.12 ±0.31,P =0.005).The relative luciferase activity in HepG2 cells co-transfected with pGL3-B7-H6 and pCMV-HBx was remarkably increased compared with the control group (17.60 ± 3.36 vs.6.73 ± 1.36,P =0.001).Western blot further demonstrated that HBx protein can significantly enhance B7-H6 protein expression.Conclusions Hepatitis B Virus X proteins enhanced B7-H6 promoter activity and promoted B7-H6 protein expression.