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
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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。Abstract: 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.
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