乙型肝炎病毒X蛋白上调白细胞介素-18基因的表达  

Study on the Up-regulation Effects of Hepatitis B Virus X Protein on Interleukin-18 Gene Expression

作　　者：刘蔚[1] 王建军[1] 成军[1] 张连峰[1] 纪冬[1] 刘妍[1] 郭江[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心,北京100039

出　　处：《中西医结合肝病杂志》2004年第3期145-148,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：国家自然科学基金资助 (No:C39970 674;No .CO30 1 1 4 0 2;No .C3990 0 1 30 ;No.C30 0 70 689)

摘　　要：目的 :了解乙型肝炎病毒 (HBV )X蛋白 (HBxAg)与白细胞介素 18(IL 18)基因表达的关系 ,研究HBxAg在HBV致病的分子生物学机制中的作用。 方法 :聚合酶链反应 (PCR)扩增人IL 18基因启动子DNA ,命名为IL 18P。以T A克隆法 ,将IL 18P基因片段连入载体pGEM T。将获得的质粒 pGEM T IL 18P ,与报告质粒pCAT3 basic分别用KpnⅠ和BglⅡ双酶切后构建IL 18启动子报告基因表达载体 pCAT3 IL 18P ,以重组表达质粒pCAT3 IL 18P和HBxAg表达载体 pcDNA HBxAg瞬时转染HepG2细胞 ,以转染 pCAT3basic的HepG2细胞为阴性对照 ,48小时后收获细胞。用酶联免疫吸附法 (ELISA)检测细胞中氯霉素乙酰转移酶 (CAT)的表达活性 ,以了解乙型肝炎病毒X蛋白对IL 18基因表达的影响。结果 :构建的报告基因表达载体 pCAT3 IL 18P经过序列分析和酶切鉴定正确。真核表达载体pcDNA3 1(阴性 ) X和 pCAT3 IL 18P共转染的HepG2细胞的CAT表达活性是pCAT3空载体的 6 2倍 ,是 pCAT3 IL 18P的 1 17倍。 结论 :乙型肝炎病毒X蛋白可上调IL 18启动子的转录活性 ,促进IL 18基因表达。Objective:To investigate the relationship of hepatitis B virus X protein and interleukin-18(IL-18) gene and explain the molecular biological mechanisms of hepatitis B virus X protein in gene expression regulations.Methods:Polymerase chain reaction (PCR) technique was employed to amplify the sequence of IL-18 promoter DNA using HepG2 cell genomic DNA astemplate, the DNA fragment was named IL-18P,and the PCR product was cloned into pGEM-T vector. The IL-18P gene was cut from pGEM-T-IL-18P by KpnⅠ and BglⅡ, and then was cloned into pCAT3 basic, the resulted vector was named pCAT3-IL-18P. pCAT3-IL-18P was transfected into the HepG2 cell line and cotransfected HepG2 cells with hepatitis B virus X protein expression vector by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-basic was used as negative control. The activity of chloramphenicol acetyltransferase(CAT) in HepG2 cells transfected was detected by an enzyme-linked immunoassay (ELISA) kit after 48 hours, which reflected the transactivating function of hepatitis B virus X protein to IL-18 gene gromoter.Results:The expressive vector pCAT3-IL-18P has been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells transfected with pCAT3-IL-18P and stimulated with pcDNA3.1(阴性)-X was 6.2 times as higher as that of pCAT3-basic, and 1.17 times as higher as that of pCAT3-IL-18P only.Conclusion:It is suggested that hepatitis B virus X protein can up-regulate IL-18 gene promoter. These results provide a new evidence to explain the molecular biological mechanisms of IL-18 in the interactions between HBV and hepatocyte.

关 键 词：乙型肝炎病毒 X蛋白 HBV HBXAG 上调白细胞介素-18 基因表达 IL-18 基因片段 

分 类 号：R346[医药卫生—基础医学]