【作者】 李志杰；

【导师】 丁壮；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 本研究分离并鉴定了一株猪繁殖与呼吸综合征病毒(PRRSV),即猪蓝耳病病毒,经病毒生物学特性测定、病毒基因鉴定、血清学试验,确定该分离毒株为美洲型PRRSV,将其命名为JL/07/SW株。根据美洲型猪繁殖与呼吸综合征病毒VR-2332株及变异毒株的核苷酸序列,针对N基因、Nsp2变异序列、以及GP5基因设计合成了的特异引物,扩增出正确的目的基因序列,测定序列经比对后,发现该毒株与美洲型毒株的同源性高于欧洲型,故判断该毒株属于美洲型变异毒株,本分离毒株其基因组在Nsp2上缺失了30个氨基酸,而GP5和M基因也存在变异,但是相对保守。膜蛋白质组是指在特定条件和特定时间细胞中的全部膜蛋白。研究膜蛋白质组的目的在于:更加深入理解膜蛋白在基本生物学过程中的作用;揭示特定细胞类型或病原体细胞表面蛋白质的表达谱,为研究疫苗和生物医学治疗积累数据。本研究应用2D-PAGE及MALDI-TOF MS/MS分析猪繁殖与呼吸综合征病毒体外感染MARC-145细胞及体内感染猪肺泡巨噬细胞(PAMs)差异细胞膜蛋白,揭示PRRSV感染引起的细胞膜蛋白表达变化,筛选并鉴定差异表达膜蛋白,并对部分蛋白进行初步的功能分析鉴定。首先用病毒体外感染MARC-145细胞,经间接免疫荧光试验确定膜蛋白提取时间为病毒感染细胞后48小时,收集约108个病毒感染及正常细胞,加入细胞膜蛋白裂解液,离心后收取上清液作为膜蛋白样品,用BCA法测定膜蛋白浓度后,进行二维凝胶电泳、染色、图像扫描与分析,然后对所选取的差异表达蛋白进行胶内酶解及质谱分析,最后进行Western Blotting分析验证二维凝胶电泳的分析结果,结果共鉴定了12个差异表达蛋白(7个上调蛋白,6个下调蛋白)。同时,本研究进行了体内试验,病毒感染仔猪,在病毒感染急性期时,采取猪臂动脉丛放血致死,获取猪肺泡巨噬细胞,进行二维凝胶电泳及质谱技术鉴定差异蛋白、Western Blotting验证,成功鉴定了17个差异表达蛋白(10个上调蛋白,7个下调蛋白)。其中波形蛋白(vimentin)、丝切蛋白( cofilin)、抗氧化应激蛋白过氧化物酶-6 (PRDX6)、抗氧化应激蛋白过氧化物酶-2 (PRDX2)、热休克蛋白(HSP)、ubiquitin(泛素)等蛋白在感染细胞中差异表达,这些蛋白可能参与了病毒感染、细胞凋亡及机体免疫等过程。对病毒感染的差异膜蛋白vimentin、PRDX2,设计特异引物进行RT-PCR扩增目的片段,构建真核表达质粒,转染细胞,筛选阳性克隆细胞,构建过表达MARC-145细胞,对构建的过表达细胞接种病毒,同时设置正常细胞对照,通过观察细胞病变、测定病毒滴度及实时荧光定量PCR方法检测病毒复制情况。结果表明过表达波形蛋白的MARC-145细胞接种PRRSV后,病毒滴度提高,荧光信号强度增加,病毒GP5基因拷贝数增加,说明波形蛋白可能是病毒受体复合物的部分,在病毒的增殖过程中起到了重要作用。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases of swine, frustrating challenge to the global swine industry. It is characterized by severe reproductive failure in sows and respiratory distress in growing pigs and piglets. The causative agent of PRRS, porcine reproductive and respiratory syndrome virus (PRRSV), is a member of the family Arteriviridae, which is composed of a group of positive sense, single stranded RNA viruses, including simian hemorrhagic fever virus (SHFV), equine arteritis virus (EAV), and lactate dehydrogenase-elevating virus (LDV). PRRSV has a highly restricted cell tropism both in vivo and in vitro. PRRSV infects the African green monkey kidney cell line, MA-104 and its derivatives, MARC-145, and CL-2621 in vitro. PRRSV preferentially infects the cells of the monocyte/macrophage lineage, especially porcine alveolar macrophages (PAMs), in the natural host. Both in PAMs and in the monkey kidney-derived cell lines, the virus enters through a mechanism of receptor-mediated endocytosis.Little is known about the molecular mechanisms of PRRSV pathogenesis. The interactions between the virus and host cell are complex and mutual when a virus invades the host. However, most of the cellular functions altered after PRRSV infection are still unknown; hence, a comprehensive study of the interaction between PRRSV and its cells was needed.The proteomic analysis of host cellular responses to virus infection may provide new insight into cellular mechanisms involved in viral pathogenesis.To date, proteomic approaches, coupling two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), have been widely used to study the mechanisms of viral infection through the comparative analysis of cellular protein profiles. This procedure, by comparing protein expression patterns of normal and infected cells, can provide exclusive information about how host cells respond to viral infection. Proteomic changes in infected host cells have been studied for many pathogenic mammal viruses, including human immunodeficiency virus type-1 (HIV-1), severe acute respiratory syndrome (SARS)-associated coronavirus, rabies virus, Nipah virus and African swine fever virus.Viral infections usually result in alterations in the host cell proteome which determine the fate of the infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus-infected pulmonary alveolar macrophages (PAMs) and MARC-145 cells, a proteomic analysis was conducted using 2-DE followed by MALDITOF-MS/MS identification. Altered expression of 44 protein spots in infected cells were identified in 2D gels, with 29 of these being characterized by MALDI-TOF-MS/MS, including 17 up-regulated proteins and 12 down-regulated proteins. Western-blot analysis confirmed the up-regulation of heat shock 27 kDa protein (HSP27), vimemtin and down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected MARC-145 cells by proteomics and provide valuable information for better understanding the function alterations of MARC-145 cells induced by PRRSV infection. The altered expression of these proteins provides a response profile of host cells (PAMs and MARC-145 cells) to PRRSV infection. Further study of these altered proteins may facilitate understanding the mechanisms of PRRSV infection and pathogenesis.Of the altered cytoskeletal proteins, cofilin-1 and vimentin were up-regulated. Maybe the cytoskeleton proteins collapse and disperse in PRRSV-infected cells. Recent evidences demonstrate that various viruses manipulate and utilize the host cytoskeleton to promote viral infection. Although these proteins may not be specific to PRRSV, most of the cytoskeleton alterations detected in PRRSV-infected cells were caused by PRRSV infection. Vimentin is an important cellular cytoskeleton component. It is in radial arrangement in cytoplasm, forming a frame to support the actomyosin system and other organelles. It can prevent the cell from being injured by changing its tension, thus keeping the cellular shape. In some viral infections, vimentin has been reported to be reorganized probably lead to unstable cytoskeletal structure. Several studies have shown that human immunodeficiency virus type 1 protease cleaves intermediate filament (IF) vimentin and induces the collapse of vimentin in infected cells. Proteomic analysis of CEFs infected with IBDV also found many cytoskeleton proteins have been up- or down-regulated significantly, vimentin protein changes at equal pace with our date. Further more, the result is the same as Fatemel’s study, they found vimentin was up-regulated in CVS infected BHK-21 cells. Therefore, precise role of vimentin cleavage and specific rearrangement of cell architecture during PRRSV infection could be important for better understanding the PRRSV replication process.Another group of up-regulated proteins of interest in our present study was ubiquitin-proteasome pathway (UPP), in the ubiquitin–proteasome pathway, a major intracellular protein degradation pathway, has recently been implicated in viral infections, including avoidance of host immune surveillance, viral maturation, viral progeny release, efficient viral replication, and reactivation of virus from latency. In this study, ubiquitin was identified as differentially expressed cellular proteins during PRRSV infection in MARC-145 cell. The best known function of ubiquitin in proteolysis is to serve as a signal for the target protein to be recognized and degraded in the proteasome. Host responses to viral infection may reflect a host defense mechanism. However, improper host protein regulation, for instance, aberrant host protein degradation, may further promote viral replication and cause tissue damage.Two antioxidative stress proteins, peroxiredoxin-6 (Prx6) and peroxiredoxin-2 (Prx2), were up-regulated, which may indicate that PRRSV infection induces host cell oxidative stress. The peroxiredoxin (Prx) family has attracted recent attention in terms of the potential antioxidant and redox signaling functions of these enzymes. Six peroxiredoxins (Prxs 1 through 6) have been identified in mammals. The cytosolic Prx2 has two highly conserved cysteine residues that become oxidized by hydrogen peroxide (H2O2) and hydroperoxides, resulting in the reversible formation of a homodimer. Human erythrocyte Prx2 reacts extremely rapidly with H2O2, with a rate constant similar to those of the catalases and glutathione peroxidases. Prx6 is a bifunctional 25 kDa protein with both GSH (glutathione) peroxidase and phospholipase A2 activities. Overexpression of Prx6 in cells can protect against oxidative stress, whereas antisense treatment results in oxidant stress and apoptosis . In this study, heat shock protein has been found both in MARC-145 cells and PAMs infected with PRRSV. Up-regulated HSPB1 has been found in cells infected with African swine fever virus, enterovirus 71, or FHV. Heat shock protein 27 KDa (HSP27) is a stress-inducible ubiquitous cellular protein and is involved in thermotolerance, cellular proliferation and apoptosis, and molecular chaperoning. It has been found to be up-regulated during certain viral infections. In the present study, heat shock protein was up-regulation during PRRSV infection, and further investigation will be required to understand its role in this. The phosphorylated HSPB1 was shown to prevent F-actin depolymerization by regulating microfilament dynamics and stabilizing the actin cytoskeleton under stress condition and to prevent caspase-independent apoptosis.We found increased intracellular levels of cystatin B in PRRSV-infected PAMs when compared with uninfected cells. A previous report has suggested that Cystatin B was even higher in both uninfected and HIV-infected MDM when compared to uninfected and HIV-infected placental macrophages where HIV replication is restricted compared to MDM.Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, including protein catabolism, antigen processing, inflammation, dystrophy and metastasis, control of their enzymatic function by cystatins is of cardinal importance. The precise roles of increased expression of this protein during PRRSV infection need further investigation.Virus induced oxidative stress is associated with the activation of phagocytosis and the release of reactive oxygen species (ROS) that plays the positive modulatory role in immune activation, eradication of viral infection and immune-induced cellular injury. Superoxide dismutase 1 (SOD1) is localized both in the cytoplasm and in the intermembrane space of mitochondria. Because of its ability to scavenge superoxide, SOD1 is considered an essential defence against the downstream generation of ROS other than O2?, even more toxic than superoxide itself. In this function, SOD1 is more efficient than SOD2. SOD1 has been found to be up-regulated during PRRSV infection in PAMs. In the present study, however, SOD1 was more notable for its down-regulation during PRRSV infection, and further investigation will be required to understand its role in this.In this experiment, Galectin-1 shows down-regulation in PRRSV infected PAMs and MARC-145 cells comparison with uninfected cells. One of such lectin families, galectins, has been recently suggested to play functional roles in various immune response processes through binding to host surface glycoproteins. All of them lack typical transmembrane segments, secretion signal peptides, and most acquire an acetylated amino terminus. Several lines of evidence imply that galectins participate in the immune response, both as immunomodulators and molecules that facilitate pathogen-host cell interactions. In addition, recent works suggest that galectins could facilitate pathogen internalization in phagocytic cells, such as macrophages. Galectin-1, a homodimer of 14 kDa subunits possessing two CRDs, recognizes the structural motif Galβ1-4GlcNAc. Because such sequences are present on cell surface glycoconjugates, galectin-1 mediates cellular adhesion processes. Furthermore, galectin-1 has been shown to have an immunoregulatory function. Administration of galectin-1 prevented experimental induced autoimmune encephalomyelitis in rats and was prophylactic and therapeutic in a rabbit model of autoimmune myasthenia gravis.Galectin-1 expression has been reported in thymus and lymphoid parenchymal epithelial cells, endothelial cells, trophoblasts, activated T cells, macrophages, activated B cells, follicular dendritic cells and CD4+CD25+ regulatory T cells. Galectin-1 can increase adsorption of X4-using isolates of HIV-1 onto CD4+ T lymphocytes, thus enhancing the overall infection process. It means that when PRRSV is infected by PAMs, it inhibits the expression of Galectin-1, and then adjusts the reproduction of the cells and resists the cell apoptosis, which is good for the persistent infection of the cells.In summary, this study adopted a gel-based proteomics approach to probe the serially changed proteins in PRRSV infected PAMs and MARC-145 cells. It is noteworthy that the comparative proteomics approach allowed for the initial identification of 29 altered cellular proteins during PRRSV infection and showed that most of the altered cellular proteins appear to have roles in revealing the viral pathogenesis. It should be noted that this study has examined only the changed proteins in PRRSV infected PAMs and MARC-145 cells. The results do not imply mechanisms of PRRSV infection and pathogenesis and the function of these altered proteins during PRRSV infection. Clearly further large scale studies are necessary to understand the roles of the differentially expressed cellular proteins in PRRSV infection.更多还原