【作者】 孙金福；

【导师】 涂长春；

【作者基本信息】 吉林大学 ， 预防兽医学， 2008， 博士

【摘要】 本研究首次用2-DE、2D DIGE和质谱鉴定等高通量蛋白质组学技术对感染CSFV的体外PK-15细胞、体内靶细胞——外周血单核细胞以及感染猪血清进行了比较蛋白质组学分析,获得了迄今最为完善的CSFV感染宿主细胞、宿主血清差异表达蛋白质组数据。CSFV感染的PK-15细胞蛋白质组分析鉴定了21个差异表达蛋白,这些蛋白中膜联蛋白(annexin 2)、不均一核内核糖核酸蛋白(hnRNPs)、翻译延长因子1δ(EF-1δ)等蛋白在感染细胞中上调表达,根据文献报道推测这些蛋白参与了CSFV的感染、基因组复制、病毒粒子的包装、出芽等生命循环过程;参与细胞凋亡激活途径的糖酵解酶甘油醛-3-磷酸脱氢酶(GAPDH)下调表达、具有抗氧化应激和抑制细胞凋亡功能的糖酵解酶磷酸甘油酸变位酶1(PGAM1)和抗氧化应激蛋白过氧化物酶-6 (PRDX6)、硫氧还蛋白(thioredoxin- like)上调表达,这些变化抑制了PK-15细胞的凋亡,可能是PK-15细胞感染CSFV不产生细胞病变的机制之一。CSFV感染的体内靶细胞——外周血单核细胞的差异蛋白质组分析鉴定了34种差异表达蛋白。7个不同的蛋白点均鉴定为细胞骨架蛋白——肌动蛋白(actin),4个不同的蛋白点鉴定为膜联蛋白annexinA1,另有膜突蛋白(moesin)、粘着斑蛋白(Vinculin)、踝蛋白(talin)等细胞骨架蛋白发生上调或下调的表达变化,这表明CSFV感染诱导了细胞骨架的解聚、降解和紊乱失调。据文献报道,细胞骨架的紊乱可诱导细胞凋亡。此外,具有激活细胞凋亡途径功能的GAPDH上调表达,抗氧化应激反应蛋白过氧化物酶(Prx-1)下调表达。这些蛋白的调节变化显示CSFV感染可能通过多种途径诱导了外周血淋巴细胞的凋亡;与细胞免疫相关的血小板反应素1(Thrombospondin 1,TSP-1)下调表达,TSP-1参与免疫功能的调节,比如,TSP-1可调节T细胞行为并参与炎性T细胞的激活和克隆增殖。TSP-1的下调表达可能是CSFV诱导免疫抑制的机制之一。该研究还检测了CSFV石门株感染诱导外周血CD4+和CD8+ T淋巴细胞亚群变化动力学。此外,用2D DIGE对感染CSFV猪血清进行了差异蛋白质组分析,鉴定了14种差异表达蛋白,其中的急性期蛋白接联珠蛋白(haptoglobin)、补体成分C4a、载脂蛋白A-1(apolipoprotein A-1)已被鉴定为其它疾病如乙型肝炎病毒(HBV)或丙型肝炎病毒(HCV)感染或相关癌症的潜在生物标记。该研究鉴定的这些CSFV感染血清差异表达蛋白具有作为CSFV感染生物标记的潜在可能性,当然,这种可能性仍需和其它动物病毒感染性疾病进行比较和评估。本研究鉴定的大量差异表达蛋白体现着宿主细胞对CSFV感染的总体反应机制,为发现新的诊断、预防和治疗靶标以及揭示CSFV致病机制奠定了基础。更多还原

【Abstract】 Classical swine fever virus (CSFV), is a small, enveloped virus with a non-segmented positive-stranded RNA genome, belonging to the genus Pestivirus within the family Flaviviridae. The viral genome is approximately 12.5 kb in size and contains a single large open reading frame (ORF). This ORF is translated into a polyprotein which is further cleaved into 11-12 mature proteins : NH2–Npro–C–Erns–E1–E2–p7–NS2–NS3–NS4A–NS4B–NS5A–NS5B–COOH. CSFV is the pathogen of classical swine fever (CSF), a highly contagious swine disease with high morbility and mortality, featuring symptoms of hemorrhagic fever and immune suppression. The disease is a notifiable one of the World organization for animal health (OIE), usually leading to substantial economic losses to the pig industry worldwide. In recent years, CSF with lower morbility and mortality and mild, chronic, atypical symptoms was often observed, even in immune pigs herd.At present, nothing is known about the interaction between CSFV and host cells. To uncover cellular protein responses in CSFV-infected PK-15 cells, a differentially proteomic analysis was conducted using 2D electrophoresis followed by MALDI-TOF-MS/MS identification. Altered expression of 35 protein spots with at least 1.5-fold quantitative differences in expression in infected cells at 48 p.i. were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 16 upregulated proteins and 5 downregulated proteins. Western-bloting analysis confirmed the upregulation of annexin 2 and downregulation of GAPDH. The altered proteins could be sorted into 7 groups according to their cellular function: cytoskeleton, energy metabolism, replication/transcription and translation processes, protein processing, anti-oxidant stress, heat shock protein and signal transduction. The altered expression of these proteins provides a response profile of PK-15 host cells to CSFV infection. Further study on these altered proteins may facilitate uncovering the mechanisms of CSFV infection and pathogenesis.According cellular function of differentially expressed proteins, it was speculated that annexin 2, hnRNPs and EF-1δmight be play critical roles in CSFV infection, genome replication, virion assembly and egress. In CSFV infected samples, GAPDH with function of apoptosis activation were down-regulated, and PGAM1 and anti-oxidative proteins (PRDX6 and thioredoxin- like) with function of apoptosis inhibition were up-regulated, which may be one mechanism of CSFV inhibiting CPE of infected PK-15 and establishing persistent infection.CSFV has particular affinity to tissue macrophage and endothelial cell. Studies indicats that CSFV infection lead to leucopenia and immune suppression. To uncover the effects on immune cells following experimental infection with CSFV Shimen strain and the mechanism of response to CSFV infection , changes of kinetics of CD4+ and CD8+ T cell subpopulation and leukocytes apoptosis in peripheral blood was analyzed using flow cytometry. Results showed that CD4+ and CD8+ T cell subpopulations were significantly reduced, the percentage of CD4+ T cell subpopulation decreased from 26.87% prior to infection to 8.83% 4 days post-infection (dpi) and 13.86% 7 dpi, respectively, and CD8+ T cell subpopulation from 28.17% prior to infection to 14.19% 4 dpi and 17.55% 7 dpi, respectively. Numbers of apoptotic leukocytes and early apoptotic leukocytes in peripheral blood of the infected pigs were significantly more than those of the control pigs, which indicated that cells apoptosis induced by CSFV infection is the major cause of leucopenia and immune suppression. Proteomic analysis indicated that 73 differentially expressed protein spots were found in CSFV-infected PBMC samples, with 55 of these(37 upregulated proteins, 18 downregulated proteins) being characterized by MALDI-TOF-MS/MS, reprensentive 34 unique proteins. Western-blot analysis indicated that Annexin A1 was remarkbaly upregulated and cofilin was downregulated, which confirmed the data of 2DE. These altered proteins are involved in cellular function of cytoskeleton, energy metabolism, nucleic acid/protein processing and anti-stress. These data provides the changes in profile of protein expression and physiological functions in PBMC from CSFV-infected porcine, which provides basis for further uncovering mechanism of CSFV pathogenesis.According cellular function of differentially expressed proteins, it was speculated that moesin, EF-1α, Bip protein might be involved in the mechanism of CSFV infection and replication. Derangement and disruption of cytoskeleton of PBMC induced by CSFV infection and upregulation of GAPDH may active apoptosis pathway of PBMC. Apoptosis of PBMC and down-regulation of T lymphocyte immune regulation factor of TSP-1 may be tightly relate to mechanism of immune suppression induced by CSFV infection.To found relevant biomarker and proteins of CSFV infection in serum from infected porcine, differentially proteomic analysis was performed using 2D-DIGE followed by MALDI-TOF-MS and RP-HPLC-MS/MS identification. Results showed that 17 differentially expressed proteins were found in CSFV-infected serum, including upregulated 9 and downregulated 8 spots. 14 of them were identified by MALDI-TOF-MS and RP-HPLC MS/MS. These proteins embodies pathological response of CSFV-infected porcine, further study is needed for potentiality of these proteins as biomarker of CSFV infection.In summary, the present study provided the first proteomic analysis of CSFV infection, and established the most comprehensive differential proteomic index of CSFV-infected cells in vitro and vivo, and CSFV-infected porcine serum. Further functional investigation of these altered proteins may facilitate understanding the pathogenic mechanisms and molecular responses of host cell to CSFV infection. This may also permit identification of biomarkers of CSFV infection and therapeutic targets or development of new diagnostic methods.更多还原