基因表达谱芯片技术筛选乙型肝炎病毒DNA多聚酶P结构域反式调节基因  

Screening and identification of genes transactivated by the Pdomain protein of hepatitis Bvirus DNA polymerase using cDNA microarray

作　　者：陈国凤[1] 王琳[1] 刘妍[1] 成军[1] 张健[1] 邵清[1] 张玲霞[1] 李莉[1] 

机构地区：[1]解放军302医院传染病研究所基因治疗研究中心全军病毒性肝炎防治研究重点实验室,北京100039

出　　处：《军医进修学院学报》2005年第2期107-109,共3页Academic Journal of Pla Postgraduate Medical School

基　　金：国家自然科学基金攻关项目(C03011402;C30070689);军队"九五"科技攻关项目(98D063);军队回国留学人员启动基金项目(98H038);军队"十五"科技攻关青年基金项目(01Q138);军队"十五"科技攻关面上项目(01MB135)

摘　　要：目的:应用基因芯片技术,对乙型肝炎病毒DNA(HBVDNA)聚合酶P结构域蛋白/逆转录酶(PR)的基因表达谱进行分析,探索PR对肝细胞基因表达的调节机制及其生物学功能。方法:设计并合成PR基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增PR蛋白编码基因片段,以常规的分子生物学技术将获得的PR编码基因片段克隆到TA载体中进行核苷酸序列的测定,构建真核表达载体pcDNA3. 1(-) PR。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pcDNA3. 1(-)的HepG2细胞进行DNA芯片分析。结果:基因芯片技术所检测的1 152个基因表达谱的筛选中, 79条基因表达水平上调, 90条基因表达水平下调。结论:应用基因表达谱芯片成功筛选了HBVDNA聚合酶PR转染细胞后差异表达基因,为进一步阐明PR蛋白可能的分子生物学机制提供依据。Objective:To study the difference in gene expression in human hepatoblastoma cell line HepG2 cells transfected with PR of hepatitis B virus DNA polymerase expressing plasmid by cDNA microarray assay and further elucidate its molecular biological function.Methods:Sequence specific primers were designed and synthesized and the PR coding DNA fragment was amplified with polymerase chain reaction (PCR) technique. The expressive vector of pcDNA3.1(-)-PR was constructed by routine molecular biological methods. cDNA microarray technology was employed to detect the mRNA from the HepG2 cells transfected with pcDNA3.1(-)-PR and pcDNA3.1(-), respectively using lipofectamine. Results:The scanning results indicate that among 1 152 genes which were gotten from gene expression profile analysis, there were 79 genes were up-regulated and 90 genes were down-regulated in PR-expressing HepG2 cells.Conclusion:cDNA microarray technology was successfully used to screen the genes differentially expressed in PR-expressing HepG2 cells, which brought some new clues for studying the potential molecular mechanism of PR of protein.

关 键 词：PR蛋白 乙型肝炎病毒DNA 反式调节基因 多聚酶 基因表达谱芯片技术 HBVDNA 基因芯片技术 结构域 基因表达水平 PCDNA3 

分 类 号：R512.62[医药卫生—内科学] R737[医药卫生—临床医学]