基因表达谱芯片技术筛选XTP4基因转染细胞差异表达基因

Screening of genes differentially expressed in HepG2 cells transfected with gene 4 transactivated by hepatitis B virus X protein (XTP4) using DNA microarray

目的 应用基因芯片技术,检测乙型肝炎病毒(HBV)X蛋白(HBxAg)反式激活基因XTP4的表达对肝母细胞瘤细胞系HepG2基因表达谱的影响,进一步阐明XTP4蛋白可能的分子生物学功能。方法 设计并合成XTP4基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增XTP4基因片段,以常规的分子生物学技术将获得的XTP4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(-)-XTP4。以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析。结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误。提取高质量的mRNA,逆转录为cDNA,进行cDNA芯片分析。在1152个基因表达谱的筛选中,发现有21个基因表达水平显著上调,38个基因表达水平显著下调。结论 应用基因表达谱芯片成功筛选了XTP4转染细胞后差异表达基因,为进一步阐明XTP4蛋白可能的生物学功能提供依据。

Objective To study the differences in gene expression in human hepatoblastoma cell line HepG2 cells transfected with XTP4-expressing plasmid and further elucidate its potential molecular biological function. Methods Sequence specific primers were designed and synthesized and the XTP4 DNA fragment was amplified with polymerase chain reaction (PCR) technique. The expressive vector of pcDNA3.1 ( - )-XTP4 was constructed by routine molecular biological methods, cDNA microarray technology was employed to detect the mRNA from the HepG2 cells transfected with pcDNA3.1 ( - )-XTP4 and pcDNAS . 1 ( - ) using lipofectamine, respectively. Results The expressive vector has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. High quality mRNA and cDNA have been prepared and successful microarray screening has been conducted. The scanning results indicate that among 1 152 genes which were obtained from gene expression profile analysis, there were 59 differences in which 21 genes were up-regulated and 38 genes were down-regulated in XTP4-expressing HepG2 cells. These genes differentially regulated by XTP4 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 XTP4-expressing HepG2 cells, which brought some new clues for studying the potential molecular mechanism of XTP4 protein.