乙型肝炎病毒表面抗原基因启动子Ⅰ结合蛋白1反式调节基因的筛选

Screening of genes differentially expressed in HepG2 cells transfected with SBPl expressing plasmid DNA using DNA microarray

目的 应用表达谱基因芯片技术 ,研究乙型肝炎病毒 (HBV)表面抗原基因启动子DNA结合蛋白 1(SBP1)反式调节基因 ,阐明SBP1蛋白可能的分子生物学功能。方法 设计并合成SBP1基因序列特异性的引物 ,应用聚合酶链反应 (PCR)技术扩增SBP1基因片段 ,以常规的分子生物学技术将获得的SBP1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(-) SBP1。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 115 2个基因容量的表达谱芯片的筛选中 ,发现有 12个基因表达水平显著上调 ,6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SBP1转染细胞后差异表达基因 ,为进一步阐明SBP1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 SBP1_expressing plasmid DNA and further elucidate its potential molecular biological function. Methods Sequence specific primers were designed and synthesized and the SBP1 DNA fragment was amplified with polymerase chain reaction (PCR) technique. The expressive vector of pcDNA3.1(-)_SBP1 was constructed. The HepG2 cells were transfected with pcDNA3.1 (-) and pcDNA3.1(-)_SBP1 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 analysis. 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 18 differences in which 12 genes were up-regulated and 6 genes were down_regulated in SBP1_expressing HepG2 cells.Conclusion cDNA microarray technology was successfully used to screen the genes differentially expressed in SBP1_expressing HepG2 cells, which bring some new clues for studying the potential molecular mechanism of SBP1 protein.