用基因芯片研究乙型肝炎病毒X蛋白羧基端缺失40个氨基酸差异表达基因_医学论文.docVIP

用基因芯片研究乙型肝炎病毒X蛋白羧基端缺失40个氨基酸差异表达基因_医学论文 用基因芯片研究乙型肝炎病毒X蛋白羧基端缺失40个氨基酸差异表达基因_医学论文 【摘要】 目的 用基因表达谱芯片技术筛选乙型肝炎病毒X蛋白全长序列和X蛋白羧基端缺失40个氨基酸序列之间差异表达的基因，初步探索HBV相关的肝细胞癌发生的分子机制。方法 将20 228条 cDNA 用点样仪点在特制的玻片上制备成表达谱芯片，将带有乙型肝炎病毒HBx蛋白全长基因序列和HBx蛋白羧基端缺失40个氨基酸（HBx3’）基因序列的pcDNA3（）的质粒分别转染肝癌细胞系Huh，提取两株细胞的总RNA，逆转录为cRNA，用Cys3和Cy5两种荧光分别标记，与表达谱芯片杂交，通过计算机扫描和数据处理，筛选出差异表达的基因。结果 差异表达的基因有167条，其中上调基因145条，下调基因22条，包括原癌及抑癌基因、细胞周期和细胞凋亡相关基因、信号转导和免疫调节相关基因、细胞增殖和代谢及基因转录等相关编码基因。结论 运用cDNA 表达谱芯片分析基因表达的差异, 能快速筛选出HBx和HBx3’之间差异表达的基因并高效率地对基因功能进行研究，为进一步研究HBV的致癌机制提供了理论依据。 【关键词】 cDNA芯片 乙型肝炎X蛋白 缺失 基因 差异表达 　　Identification of Differentially Expressed Genes in Hepatitis B Virus X Protein C 　　Abstract：Objective To investigate the differentially expressed genes of the full－length sequence of HBV X protein and the sequence of the HBV X protein deletion mutant cRNAs were isolated from Huh7 cells transfected with pcDNA3() integrated the sequcece of HBx protein cterminally truncated 40 amino cids and pcDNA3() integrated the full length sequence of HBx protein vectors, then RNAs were reversely transcripted into cRNA and hybridized onto Agilent Genechip expression microarrays containing 20228 human cDNA. Analyses were performed to determine the different pattern of gene expression in the cells with full length HBx protein and HBx protein cterminally truncated 40 amino acids and the changes in the expression level by computer. Results Through this cDNA microarray we identified 167 differentially expressed genes and among those we further identified 145 upregulated and 22 downregulated genes and two novel human genes, the existing genes including oncogenes and tumor suppression genes, cell cycle and cell apoptosis genes, signal transduction and immune regulation genes, cell proliferation and metabolism genes, and gene transcriptionrelated genes. Conclusion Microarray technique is effective in screening the differentially expressed genes and exploring their function between two different kinds of Huh7 cells transfected with pc