定量蛋白质组学技术分析猪繁殖与呼吸综合征病毒感染肺泡巨噬细胞的差异表达蛋白质

doi:10.11843/j.issn.0366-6964.2016.06.018

摘要/Abstract

摘要：

作者采用定量蛋白质组学方法，对猪繁殖与呼吸综合征病毒（PRRSV）感染猪肺泡巨噬细胞（PAMs）和正常PAMs的蛋白质组进行差异分析。通过高效液相色谱和电喷雾串联质谱仪定量分析PRRSV感染细胞与未感染对照细胞间的差异表达蛋白质，并运用Max-Quant 1.0.7.4软件结合蛋白质数据库PaxDb进行鉴定。与对照组相比，感染组共鉴定出39个差异表达蛋白质，其中30个蛋白质表达上调，9个蛋白质表达下调。通过Western blot和ELISA验证了感染的PAMs中SPP1、IFIT3、STAT3及IL-8的表达情况与质谱鉴定结果一致。通过液相色谱—串联质谱联用（LC-MS/MS）技术，筛选得到多个与PRRSV感染相关的差异表达蛋白质，为PRRSV发病的分子机制分析提供了新依据。

Abstract:

The purpose of our research was to study the differential expression proteins of Porcine reproductive and respiratory syndrome virus (PRRSV) infected porcine alveolar macrophage (PAMs) relative to the un-infected control PAMs.Differential proteins in response to PRRSV infection were analyzed by High Performance Liquid Chromatography coupled with tandem mass spectrometry，and then identified using software Max-Quant 1.0.7.4 combining with the protein database PaxDb.Thirty-nine differential proteins were identified in the PRRSV infected group，including thirty up-regulated proteins and nine down-regulated proteins.The up-regulation of SPP1，IFIT3 and IL-8，and down-regulation of STAT3 in PRRSV infected PAMs were verified by Western blot and ELISA.This study provides new evidences for further studies on the molecular mechanism of PRRSV pathogenesis by using LC-MS/MS.

中图分类号:

S858.285.3

刘龙，陈红英. 定量蛋白质组学技术分析猪繁殖与呼吸综合征病毒感染肺泡巨噬细胞的差异表达蛋白质[J]. 畜牧兽医学报, 2016, 47(6): 1222-1231.

LIU Long，CHEN Hong-ying. Analysis of Differential Expression Proteins in Porcine Reproductive and Respiratory Syndrome Virus Infected Pulmonary Alveolar Macrophage by Quantitative Proteomics[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1222-1231.

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参考文献

［1］WENSVOORT G，TERPSTRA C，POL J M，et al.Mystery swine disease in The Netherlands：the isolation of Lelystad virus［J］.Vet Q，1991，13(3)：121-130.
［2］DUAN X，NAUWYNCK H J，PENSAERT M B.Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV)［J］.Vet Microbiol，1997，56(1-2)：9-19.
［3］DING Z，LI Z J，ZHANG X D，et al.Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus［J］.Vet Immunol Immunopathol，2012，145(1-2)：206-213.
［4］LI J N，GUO D W，HUANG L，et al.The interaction between host Annexin A2 and viral Nsp9 is beneficial for replication of porcine reproductive and respiratory syndrome virus［J］.Virus Res，2014，189：106-113.
［5］LIU L，LEAR Z，HUGHES D J，et al.Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology［J］.Vet Microbiol，2015，176(1-2)：109-119.
［6］钱小红.定量蛋白质组学分析方法［J］.色谱，2013，31(8)：719-723.
QIAN X H.The analytical method of quantitative proteomics［J］.Chinese Journal of Chromatography，2013，31(8)：719-723.(in Chinese)
［7］YANG Y，AN T，GONG D，et al.Identification of porcine serum proteins modified in response to HP-PRRSV HuN4 infection by two-dimensional differential gel electrophoresis［J］.Vet Microbiol，2012，158(3-4)：237-246.
［8］ZHANG H，GUO X，GE X，et al.Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis［J］.J Proteome Res，2009，8(6)：3091-3097.
［9］李铁求，滕伊漓，邹亚光，等.基于基因芯片的前列腺癌转移高表达基因SPP1的生物信息学分析［J］.中华男科学杂志，2014，20(11)：984-990.
LI T Q，TENG Y L，ZOU Y G，et al.The highly expressed secreted phosphoprotein 1 gene in prostate cancer metastasis：A microarray-based bioinformatic analysis［J］.National Journal of Andrology，2014，20(11)：984-990.(in Chinese)
［10］ROLLO E E，DENHARDT D T.Differential effects of osteopontin on the cytotoxic activity of macrophages from young and old mice［J］.Immunology，1996，88(4)：642-647.
［11］ZHAO W，WANG L，ZHANG L，et al.Differential expression of intracellular and secreted osteopontin isoforms by murine macrophages in response to toll-like receptor agonists［J］.J Biol Chem，2010，285(27)：20452-20461.
［12］MIETTINEN J J，MATIKAINEN S，NYMAN T A.Global secretome characterization of herpes simplex virus 1-infected human primary macrophages［J］.J Virol，2012，86(23)：12770-12778.
［13］扶琼.SLE相关基因IFIT3的功能及分子机制研究［D］.上海：上海交通大学，2008.
FU Q.A study on biological functions and molecular mechanisms of a lupus related gene IFIT3［D］.Shanghai：Shanghai Jiaotong University，2008.(in Chinese)
［14］LU Q，BAI J，ZHANG L，et al.Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus［J］.J Proteome Res，2012，11(5)：2890-2903.
［15］ZHANG L，LIU J，BAI J，et al.Poly(I：C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3［J］.Antiviral Res，2013，99(3)：197-206.
［16］LEE H，HERRMANN A，DENG J H，et al.Persistently activated Stat3 maintains constitutive NF-κB activity in tumors［J］.Cancer Cell，2009，15(4)：283-293.
［17］MIZUTANI T，FUKUSHI S，MURAKAMI M，et al.Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells［J］.FEBS Lett，2004，577(1-2)：187-192.
［18］HILL E R，KOGANTI S，ZHI J，et al.Signal transducer and activator of transcription 3 limits Epstein-Barr virus lytic activation in B lymphocytes［J］.J Virol，2013，87(21)：11438-11446.
［19］HUANG S C，RAGHAVARAJU G，LIU H S.High expression of vascular endothelial growth factor in EV71-infected patients does not originate from EV71-infected cells［J］.Intervirology，2010，53(6)：394-401.
［20］SHEN S，NISO-SANTANO M，ADJEMIAN S，et al.Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity［J］.Mol Cell，2012，48(5)：667-680.
［21］STRIETER R M，CHENSUE S W，BASHA M A，et al.Human alveolar macrophage gene expression of interleukin-8 by tumor necrosis factor-α，lipopolysaccharide，and interleukin-1β［J］.Am J Respir Cell Mol Biol，1990，2(4)：321-326.
［22］BARTON G M，MEDZHITOV R.Toll-like receptor signaling pathways［J］.Science，2003，300(5625)：1524-1525.
［23］PETRY D B，LUNNEY J，BOYD P，et al.Differential immunity in pigs with high and low responses to porcine reproductive and respiratory syndrome virus infection［J］.J Anim Sci，2007，85(9)：2075-2092.
［24］LUNNEY J K，FRITZ E R，REECY J M，et al.Interleukin-8，interleukin-1β，and interferon-γ levels are linked to PRRS virus clearance［J］.Viral Immunol，2010，23(2)：127-134.

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