生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
6期
759-766
,共8页
王继峰%李靖%康晓平%吴晓燕%钱小红%应万涛%杨银辉
王繼峰%李靖%康曉平%吳曉燕%錢小紅%應萬濤%楊銀輝
왕계봉%리정%강효평%오효연%전소홍%응만도%양은휘
高致病性H5N1禽流感病毒%A549细胞%蛋白质组%稳定同位素标记氨基酸技术(SILAC)
高緻病性H5N1禽流感病毒%A549細胞%蛋白質組%穩定同位素標記氨基痠技術(SILAC)
고치병성H5N1금류감병독%A549세포%단백질조%은정동위소표기안기산기술(SILAC)
highly pathogenic H5N1 avian influenza virus%A549 cell lines%proteome%stable isotope labeling by amino acids in cell culture(SILAC)
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。
目的：鑒定高緻病性H5N1禽流感病毒感染A549肺癌細胞後，細胞蛋白質組的錶達變化，併鑒定特異分子通路的改變及其涉及的關鍵蛋白質分子。方法：利用穩定同位素標記氨基痠技術（SILAC）標記A549細胞，得到“重標”或“輕標”的A549細胞；“重標”細胞感染高緻病性H5N1禽流感病毒24 h後提取細胞總蛋白，與從未感染病毒的“輕標”細胞中提取的總蛋白等量混閤，酶解肽段，經正交反相色譜分離後用質譜鑒定，對數據進行定性和定量分析。結果：共鑒定到3504箇蛋白質，有定量信息的蛋白質為2469箇，病毒感染後錶達量升高的蛋白質為72箇，錶達量降低的蛋白質為66箇，其中包括參與多箇分子調控途徑如RNA剪接體、榦擾素誘導通路、汎素化通路、胰島素通路等的蛋白質。結論：建立瞭利用SILAC技術研究宿主細胞-病毒相互作用的方法，髮現瞭高緻病性H5N1禽流感病毒感染宿主細胞相關的關鍵蛋白質，為探索H5N1病毒緻病的分子機理提供瞭理論基礎。
목적：감정고치병성H5N1금류감병독감염A549폐암세포후，세포단백질조적표체변화，병감정특이분자통로적개변급기섭급적관건단백질분자。방법：이용은정동위소표기안기산기술（SILAC）표기A549세포，득도“중표”혹“경표”적A549세포；“중표”세포감염고치병성H5N1금류감병독24 h후제취세포총단백，여종미감염병독적“경표”세포중제취적총단백등량혼합，매해태단，경정교반상색보분리후용질보감정，대수거진행정성화정량분석。결과：공감정도3504개단백질，유정량신식적단백질위2469개，병독감염후표체량승고적단백질위72개，표체량강저적단백질위66개，기중포괄삼여다개분자조공도경여RNA전접체、간우소유도통로、범소화통로、이도소통로등적단백질。결론：건립료이용SILAC기술연구숙주세포-병독상호작용적방법，발현료고치병성H5N1금류감병독감염숙주세포상관적관건단백질，위탐색H5N1병독치병적분자궤리제공료이론기출。
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 chroma-tography 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. Conclu-sion: 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.
