用SILAC技术研究感染H5N1禽流感病毒后A549肺癌细胞蛋白质组的表达变化

Cellular Proteome Alterations in Highly Pathogenic H5N1 Avian Influenza Virus-Infected Human Lung Cell Line A549

目的:鉴定高致病性H5N1禽流感病毒感染A549肺癌细胞后,细胞蛋白质组的表达变化,并鉴定特异分子通路的改变及其涉及的关键蛋白质分子。方法:利用稳定同位素标记氨基酸技术(SILAC)标记A549细胞,得到"重标"或"轻标"的A549细胞;"重标"细胞感染高致病性H5N1禽流感病毒24 h后提取细胞总蛋白,与从未感染病毒的"轻标"细胞中提取的总蛋白等量混合,酶解肽段,经正交反相色谱分离后用质谱鉴定,对数据进行定性和定量分析。结果:共鉴定到3504个蛋白质,有定量信息的蛋白质为2469个,病毒感染后表达量升高的蛋白质为72个,表达量降低的蛋白质为66个,其中包括参与多个分子调控途径如RNA剪接体、干扰素诱导通路、泛素化通路、胰岛素通路等的蛋白质。结论:建立了利用SILAC技术研究宿主细胞-病毒相互作用的方法,发现了高致病性H5N1禽流感病毒感染宿主细胞相关的关键蛋白质,为探索H5N1病毒致病的分子机理提供了理论基础。

Objective： To determine the cellular proteome responses of human lung A549 cell lines to the highly pathogenic H5N1 avian influenza virus infection, explore changes of specific molecular pathways and identify the key proteins involved in the infection. Methods： By using stable isotope labeling by amino acids in cell culture （SILAC） method to obtain ＂heavy＂ labeled cell lines which were infected with H5N1 virus and ＂light＂ labeled cell lines which were not infected, from which the cellular proteins were extracted and mixed in even amounts. Then the peptides derived from the mixed proteins digestion were identified by orthogonal reversed-phase chromatography coupled with mass spectroscopy and performed qualitative and quantitative analysis. Results： Of the total 3504 identified proteins and 2469 proteins with quantitative information, 72 were significantly up-regulated, 66 were significantly down-regulated. These proteins were involved in several molecular regulation pathways, including RNA splicesome, interferon inducible pathways, ubiquitin degradation pathway, insulin pathway and so on. Conclusion： We successfully established a strategy to explore the virus-host cell interactions with SILAC method. The identification of the key proteins involved in highly pathogenic H5N1 avian influenza virus infection, providedthe theoretical basis forunderstanding the molecular pathogenesis of H5N1 infection.