Skiba Martin, Mettenleiter Thomas C, Karger Axel
Institute of Molecular Biology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
J Virol. 2008 Oct;82(19):9689-99. doi: 10.1128/JVI.00995-08. Epub 2008 Jul 23.
A quantitative proteome study using the stable isotope labeling with amino acids in cell culture technique was performed on bovine kidney cells after infection with the alphaherpesvirus pseudorabies virus (PrV), the etiological agent of Aujeszky's disease. To enhance yields of proteins to be identified, raw extracts were fractionated by affinity solid-phase extraction with a combination of a cibacron blue F3G-A and a heparin matrix and with a phosphoprotein-specific matrix. After two-dimensional gel electrophoresis in different pH ranges between pH 3 and pH 10, 2,600 proteins representing 565 genes were identified by mass spectrometry and screened for virus-induced changes in relative protein levels. Four hours after infection, significant quantitative variations were found for constituents of the nuclear lamina, representatives of the heterogeneous nuclear ribonucleoproteins, proteins involved in membrane trafficking and intracellular transport, a ribosomal protein, and heat shock protein 27. Several proteins were present in multiple charge variants that were differentially affected by infection with PrV. As a common pattern for all these proteins, a mass shift in favor of the more acidic isoforms was observed, suggesting the involvement of viral or cellular kinases.
运用细胞培养中氨基酸稳定同位素标记技术，对感染奥耶斯基氏病病原体——甲型疱疹病毒伪狂犬病病毒（PrV）后的牛肾细胞进行了定量蛋白质组研究。为提高待鉴定蛋白质的产量，粗提物通过用汽巴蓝F3G - A和肝素基质以及磷蛋白特异性基质组合进行亲和固相萃取来分级分离。在pH 3至pH 10的不同pH范围内进行二维凝胶电泳后，通过质谱鉴定了代表565个基因的2600种蛋白质，并筛选病毒诱导的相对蛋白质水平变化。感染后4小时，发现核纤层成分、不均一核核糖核蛋白代表、参与膜运输和细胞内运输的蛋白质、一种核糖体蛋白以及热休克蛋白27存在显著的定量变化。几种蛋白质存在多种电荷变体，它们受PrV感染的影响不同。对于所有这些蛋白质，共同的模式是观察到质量向更酸性异构体偏移，这表明病毒或细胞激酶参与其中。
