乙型肝炎病毒X蛋白对B7-H6基因的激活作用  

Hepatitis B virus X protein promotes B7-H6 gene activation

作　　者：邹勇[1] 杨小安[2] 郑常龙[3] 潘兴飞[4] 徐启桓[2] Zou Yong;Yang Xiaoan;Zhen Changlong;Pan Xingfei;Xu Qihuan(Department of Blood Transfusion, Department of Infectious Diseas;Department of Emergency, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Chin;Department of Infectious Disease, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, Chin)

机构地区：[1]中山大学附属第三医院输血科,广州510630 [2]中山大学附属第三医院感染科,广州510630 [3]中山大学附属第三医院诊科,广州510630 [4]广州医科大学附属第三医院感染科,510150

出　　处：《中华实验和临床病毒学杂志》2018年第3期255-258,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：广东省科技计划项目（2014A020212575,2016A020215215）;广东省自然科学基金（2016A030313357）

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KpnI和HindⅢ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。ObjectiveTo investigate the key factor（s） of hepatitis B virus X protein （HBx） promoting B7-H6 gene activation.MethodsThe 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 HindⅢ, 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 co-transfected 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.ResultsA 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.ConclusionsHepatitis B Virus X proteins enhanced B7-H6 promoter activity and promoted B7-H6 protein expression.

关 键 词：NKP30 B7-H6 启动子 肝炎病毒 乙型 

分 类 号：R373.21[医药卫生—病原生物学]