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目的应用基因芯片技术,筛选能被乙型肝炎病毒E抗原(HBeAg)肝细胞作用蛋白AK026018反式调节的靶基因,初步研究该蛋白的生物学功能。方法应用反转录聚合酶链反应(RT-PCR)技术,从HepG2细胞中扩增编码AK026018蛋白的全基因,构建真核表达载体,转染肝母细胞瘤系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3·1的HepG2细胞进行DNA芯片分析并比较。结果经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确。在8464个基因表达谱的筛选中,发现有122个基因有差异表达,其中78种基因表达水平显著下调,45种基因表达水平显著上调。结论成功地应用DNA芯片技术筛选出HBeAg结合蛋白新基因AK026018的反式调节蛋白,证明该基因对于肝细胞基因表达谱有显著影响。
Objective To screen the target gene transactivated by hepatitis B virus E antigen (HBeAg) hepatocyte protein AK026018 by gene chip technology and to study the biological function of this protein. Methods The whole gene encoding AK026018 protein was amplified by RT-PCR from HepG2 cells and the eukaryotic expression vector was constructed and transfected into Hepatoblastoma cell line HepG2 to extract the total mRNA and reverse transcription As a cDNA, and compared with HepG2 cells transfected with the blank expression vector pcDNA3.1. Results The constructed recombinant expression vector was identified by restriction endonuclease analysis and DNA sequencing. Of 8464 gene expression profiles, 122 genes were found to be differentially expressed, of which 78 genes were significantly down-regulated and 45 genes were significantly up-regulated. Conclusion The trans-regulatory protein of new gene of HBeAg-binding protein AK026018 was successfully screened by DNA chip technology, and the gene expression profile of hepatocyte was significantly influenced by this gene.