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目的应用基因表达谱芯片(基因芯片)技术检测乙型肝炎病毒DNA聚合酶(HBVDNAP)三个功能域[(N末端蛋白(TP),逆转录酶DNA多聚酶(PR),核糖核酸酶H(RNaseH)]的表达对肝母细胞瘤细胞HepG2基因表达谱的影响,进一步阐明HBVDNAP对肝细胞基因表达的调节机制及其生物学功能。方法以常规分子生物学技术分别构建真核表达载体pcDNA3.1()TP,pcDNA3.1()PR和pcDNA3.1()RNaseH,以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析。结果TP有111条基因表达水平上调,88条基因表达水平下调。PR有79条基因表达水平上调,90条基因表达水平下调。RNaseH有113条基因表达水平上调,109条基因表达水平下调。结论应用基因芯片成功筛选HBVDNAP三个功能域蛋白转染细胞后的差异表达基因,为进一步研究HBVDNAP的反式激活作用及免疫调节机制提供了新的依据。
Objective To detect the expression of hepatitis B virus DNA polymerase (HBVDNAP) in three functional domains [(N terminal protein (TP), reverse transcriptase DNA polymerase (PR), RNaseH )] Expression on hepatoblastoma HepG2 gene expression profile, to further clarify the regulatory mechanism of HBVDNAP on hepatocyte gene expression and its biological function.Methods Using conventional molecular biology techniques to construct eukaryotic expression vector pcDNA3.1 () TP, pcDNA3.1 () PR and pcDNA3.1 () RNaseH were transfected into hepatoblastoma cell line HepG2 by Lipofectamine 2000. The mRNA was extracted and reverse transcribed into cDNA. The recombinant plasmid was transfected into the blank expression vector pcDNA3.1 ) Of HepG2 cells were analyzed by DNA microarray.Results The expression of 111 genes in TP was up-regulated and the expression of 88 genes was down-regulated in PR.There were 79 genes were up-regulated in PR and 90 genes were down-regulated.The expression of 113 genes in RNaseH was up-regulated , 109 genes were downregulated.Conclusion The successful screening of differentially expressed genes of HBVDNAP transfected cells by gene chip provides a new basis for further study on transactivation and immunoregulatory mechanism of HBVDNAP .