应用基因表达谱芯片技术筛选XTP6基因转染细胞差异表达基因

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XTP6的表达对肝母细胞瘤系HepG2基因表达谱的影响,进一步阐明XTP6蛋白可能的分子生物学功能.方法设计并合成XTP6基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增XTP6基因片段,以常规的分子生物学技术将获得的XTP6编码基因片段克隆到TA载体中进行核苷酸序列测定,构建真核表达载体pcDNA3.1(-)-XTP6.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的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1 152个基因表达谱的筛选中,发现21个基因表达水平显著上调,18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XTP6转染细胞后差异表达基因,为进一步阐明XTP6蛋白可能的生物学功能提供依据.

Screening of genes differentially expressed in HepG2 cells transfected with XTP6 using DNA microarray

Objective To study the differences in gene expression in human hepatoblastoma cell line HepG2 cells transfected with XTP6-expressing plasmid and further elucidate its potential molecular biological function.Methods Sequence specific primers were designed and synthesized and the XTP6 DNA fragment was amplified with polymerase chain reaction (PCR) technique.The expressive vector of pcDNA 3.1-XTP6 was constructed.The HepG2 cells were transfected by pcDNA 3.1(-) and pcDNA 3.1(-)-XTP6 using FuGENE 6 transfection reagent, respectively.The mRNA was isolated and reverse transcribed. The cDNAs were subjected for microarray screening with 1152 cDNA probes.Results The expressive vector had been constructed and confirmed by restriction enzyme digestion and DNA sequencing anslysis. High quality mRNA and cDNA had been prepared and successful microarray screening was conducted. The scanning results indicated that among 1152 genes which were obtained from gene expression profile analysis,there were 39 differences in which 21 genes were up-regulated and 18 genes were down-regulated in XTP6-expressing HepG2 cells.Conclusions cDNA microarray technology was successfully used to screen the genes differentially expressed in XTP6-expressing HepG2 cells, which bring some new clues for studying the potential molecular mechanism of XTP6 protein.