应用表达谱芯片技术筛选HBeAg反式调节基因

目的 筛选与克隆HBeAg反式调节基因，了解其在体内的调节功能线索及机制。方法 以分子生物学技术构建HBeAg的真核表达载体pcDNA3．1(-)-HBeAg，转染HepG2细胞，以空载体pcDNA3．1(-)为平行对照，制备转染后的细胞裂解液，提取mRNA。应用基因表达谱芯片技术对差异表达mRNA进行检测和分析。结果 HBeAg表达质粒pcDNA3．1(-)-HBeAg经酶切鉴定和DNA测序鉴定正确。经基因表达谱芯片分析，5种基因的表达水平上调，7种基因的表达水平下调。结论 筛选到一些与细胞信号传导、免疫调节、蛋白翻译合成、肿瘤、神经系统发生相关的HBeAg反式调节的靶基因。

Screening and identification of the genes transactivated by HBeAg using microarray assay

Objective To screen and identify the up- and down-regulated genes of HBeAg in hepatocyte, and to explore the possible function of HBeAg in vivo.Methods The recombined expression plasmid pcDNA3.1(-)-HBeAg was constructed, and HepG2 cells were transfected. Total mRNA was isolated from the HepG2 cells transfected with pcDNA3.1(-) and pcDNA3- HBeAg,respectively. Microarray was employed for detecting and analyzing of mRNA from the HepG2 cells.Results The expressive vector of pcDNA3.1(-)-HBeAg has been constructed and confirmed by restriction enzyme digestion and DNA sequencing. After screening with DNA microarray, five genes were found up-regulated, and seven genes were found down-regulated.Conclusion The obtained sequences may be target genes transactivated by HBeAg, among which some genes coding proteins involved in immunoloregulation, signal transduction in cells, translation and synthesis of protein, and correlated with tumor and nervous system.