基因表达谱芯片技术筛选HBV DNA多聚酶RNase H反式调节基因  

Screening and Identification of Genes Transactivated by the RNase H of Hepatitis B Virus DNA Polymerase Using cDNA Microarray

作　　者：陈国凤[1] 王琳 成军[3] 张健[1] 刘妍[2] 刘敏[2] 张玲霞[4] 李莉[1] 

机构地区：[1]中国人民解放军第302医院感染四科,北京100039 [2]中国人民解放军传染病研究所基因治疗研究中心 [3]北京市地坛医院 [4]中国人民解放军第302医院专家组

出　　处：《中西医结合肝病杂志》2005年第2期81-84,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：国家自然科学基金攻关项目 (C0 30 1 1 4 0 2 ;C30 0 70 689) ;军队"九五"科技攻关项目 (98D0 63) ;军队回国留学人员启动基金项目 (98H0 38) ;军队"十五"科技攻关青年基金项目 (0 1Q1 38) ;军队"十五"科技攻关面上项目 (0 1MB1 35)

摘　　要：目的:应用基因表达谱芯片(基因芯片)技术,检测乙型肝炎病毒DNA聚合酶RNaseH结构域蛋白(HBVDNAP RNaseH ,RNaseH)的表达对肝母细胞瘤细胞HepG2基因表达谱的影响,进一步阐明RNaseH对肝细胞基因表达的调节机制及其生物学功能。方法:以常规的分子生物学技术构建真核表达载体pcDNA 3 1(-) RNaseH ,以脂质体转染肝母细胞瘤细胞系HepG2 ,提取mRNA ,逆转录为cDNA ,与转染空白表达载体pcDNA 3 1(-)的HepG2细胞进行cDNA芯片分析。结果:RNaseH表达质粒转染的细胞有2 2 2条差异表达基因,其中113条基因表达水平上调,10 9条基因表达水平下调。结论:应用基因芯片成功筛选了RNaseH转染细胞后差异表达基因,为进一步阐明RNaseH的反式激活作用及免疫调节机制提供了新的依据。Objective:To study the difference in gene expression in human hepatoblastoma cell line HepG2 cells transfected with RNase H of hepatitis B virus DNA polymerase(HBV DNA P)expressing plasmid by microarray assay and further elucidate its molecular biological function.Methods:Sequence specific primers were designed and synthesized and the RNase H coding DNA fragment was amplified with polymerase chain reaction (PCR) technique using G318A7 plasmid containing the full length of HBV DNA AF384372 cDNA as the template. The expressive vector of pcDNA 3.1(-)RNase H was constructed by routine molecular biological methods. cDNA microarray technology was employed to detect the mRNA from the HepG2 cells transfected with pcDNA 3.1(-)RNase H and pcDNA3.1(-), respectively using lipofectamine.Results:The expressive vector has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. High quality mRNA and cDNA were prepared and successful microarray screening was conducted. The scanning results indicate that among 1 152 genes which were gotten from gene expression profile analysis,there were 113 genes were up-regulated and 109 genes were down-regulated in RNase H-expressing HepG2 cells. These genes differentially regulated by RNase H included human genes encoding proteins involved in cell signal transduction,cell apoptosis,cell proliferation and differentiation.Conclusion:cDNA microarray technology was successfully used to screen the genes differentially expressed in RNase H-expressing HepG2 cells,which brougth some new clues for studying trans-regulated and immune regulation mechanism of RNase H.

关 键 词：基因表达谱芯片技术 DNA多聚酶 反式调节基因 筛选 RNaseH HBV 细胞系HepG2 差异表达基因 基因表达水平 肝母细胞瘤细胞 分子生物学技术 HepG2细胞 DNA聚合酶 乙型肝炎病毒 真核表达载体 pcDNA3 免疫调节机制 反式激活作用 

分 类 号：R346[医药卫生—基础医学]