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目的研究458nt~1 308nt乙型肝炎病毒剪接特异性蛋白TSR′r′对Huh7细胞基因表达的影响,探讨其可能的致病机制。方法PCR扩增TSR′r′编码序列并克隆入pcDNA3.1/HisC。以FuGENE6将重组表达载体及相应空载体分别瞬时转染Huh7细胞,Trizol抽提细胞总蛋白与总RNA;以融合表达多肽表位抗体为一抗,Western blot检测目的蛋白的表达;用Affymetrix Genechip U133 plus 2.0人类基因表达谱芯片检测Huh7肝细胞基因转录的变化,并以半定量RT-PCR进一步验证。结果成功构建重组真核表达载体pcDNA3.1/HisC-TSR′r′,转染48 h后在Huh7细胞中表达TSR′r′蛋白。TSR′r′导致Huh7细胞载脂蛋白H等27个基因转录上调,其中包括5种代谢相关基因,7种免疫相关基因,7种胶原及胞外基质基因,5种干扰素诱导蛋白基因;TSR′r′还导致Huh7细胞核受体共激活物1基因在内的7种基因转录下降。结论458nt~1 308nt乙型肝炎病毒剪接特异性蛋白可能多方面影响肝细胞功能,具有重要的致病意义。
Objective To study the effect of TSR’r ’, a splicing protein of 458nt ~ 1 308nt, on the gene expression of Huh7 cells and to explore its possible pathogenic mechanism. Methods The TSR’r ’coding sequence was amplified by PCR and cloned into pcDNA3.1 / HisC. Huh7 cells were transiently transfected with the recombinant expression vector and the corresponding empty vector with FuGENE6, and the total protein and total RNA of the cells were extracted with Trizol. The fusion protein was expressed by Western blot to detect the expression of the target protein. Affymetrix Genechip U133 plus 2.0 human gene expression microarray to detect the gene transcription of Huh7 hepatocytes, and further verified by semi-quantitative RT-PCR. Results The recombinant eukaryotic expression vector pcDNA3.1 / HisC-TSR’r was successfully constructed and expressed in Huh7 cells 48 h after transfection. TSR’r ’led to the transcriptional up-regulation of 27 genes such as apolipoprotein H in Huh7 cells, including 5 metabolic related genes, 7 immune-related genes, 7 collagen and extracellular matrix genes and 5 interferon-inducible protein genes. TSR ’R’ also led to a decrease in transcription of the seven genes, including Huh7 nuclear receptor coactivator 1 gene. Conclusion 458 ~ 1 308 nt hepatitis B virus splicing specific protein may affect liver cell function in many aspects, which has important pathogenic significance.