猪圆环病毒2型-猪瘟病毒共感染细胞的蛋白质组研究

发布时间：2018-04-20 02:35

  本文选题：猪圆环病毒2型 + 猪瘟病毒　； 参考：《浙江大学》2016年博士论文

【摘要】：随着规模化养殖技术的不断进步和发展、交通运输的快捷等因素,增加了猪群之间的接触机会,也为病原体的流行和传播创造了机会,造成发病猪群呈现两种或多种病原的混合感染的现状。越来越多的研究表明PCV2和CSFV在猪体内能够发生共感染,并且临床的研究表明PCV2能干扰CSFV弱毒疫苗的保护效力,给养猪业带来重大的经济损失。然而,PCV2-CSFV共感染时相互作用和对宿主的影响机制还未知。在本研究中,为获得效价较高的CSFV C株的多克隆抗体,利用PEG6000浓缩法和蔗糖密度梯度离心纯化法,制备纯化的CSFV样品。结果表明,经浓缩纯化的CSFV仍具有较高的感染活性,且毒价为病毒原液的101.5倍以上。将纯化的CSFV作为抗原接种新西兰大白兔,获得特异性和效价均较高的抗CSFV兔多抗血清。为了有效地研究PCV2和CSFV共感染过程中病毒的交互影响以及细胞参与病毒复制的潜在机制,建立了一个的体外共感染模型。在该模型下PCV2不受CSFV复制影响,CSFV对PCV2没有免疫排斥现象;而CSFV的增殖却以PCV2剂量依赖性的方式降低。PCV2和CSFV能定位于同一个细胞,且CSFV的E2从正常的细胞质定位,易位到细胞核中。此外,相同剂量的PCV2感染PK15和PK15-CSFV,PCV2的感染率相同,并且介导的细胞凋亡程度无显著性差异;然而,PCV2剂量与细胞凋亡呈现正相关,表明PCV2介导的凋亡可能与CSFV复制下调有关。另外,灭活PCV2及PCV2病毒成分孵育细胞不能介导细胞凋亡或影响CSFV复制,表明PCV2复制介导的凋亡影响CSFV复制。这些结果足以解释临床上PCV2和CSFV共同感染导致更严重的临床症状,以及CSFV弱毒疫苗免疫失败问题。为了探究在PCV2和CSFV共感染过程中涉及的宿主因子和分子机制,使用iTRAQ标记结合LC-MS/MS质谱鉴定技术,分析阴性(NE)、PCV2单独感染(SP)、CSFV单独感染(SC)和PCV2-CSFV共感染(PC)的PK15样品的整体蛋白质组表达情况。在3次重复实验中,共有3932个蛋白均检测到。以表达比例≥ 1.2或≤0.83且p 5 0.05作为上下调的阈值,与NE相比,SP中上、下调蛋白数分别为304个和198个,SC中分别为60个和61个,PC中分别为196个和156个。相对定量PCR、蛋白免疫印迹和共聚焦激光扫描显微镜技术验证的结果表明,大部分基因/蛋白的表达趋势与iTRAQ蛋白质组结果一致。聚类、GO和KEGG分析证明在PCV2和CSFV共感染过程中PCV2具有主导作用。对PC/SC差异表达蛋白进行IPA分析,结果显示蛋白14-3-3ζ、cullin3、ERK1/2、caspase和NF-κB可能在PCV2影响CSFV在体外完成生命周期的过程中具有重要作用。另外,进一步对共感染条件下特异性差异表达的184个蛋白进行KEGG和IPA分析,结果表明线粒体呼吸链复合物I、IV和V的多个蛋白均下调表达,由此推断线粒体可能是PCV2和CSFV共感染时的主要靶标;除了线粒体功能失常外,共感染还特异性影响了氧化磷酸化、Nrf2介导的氧化应激、tRNA装载、Myc介导的凋亡信号传导和细胞内多种代谢通路的信号通路。这些数据为研究PCV2和CSFV共感染过程对细胞通路的影响及其分子机制提供参考。综上,本研究获得了高效价高纯度的CSFV C株病毒以及抗CSFV的兔多抗,为检测CSFV提供工具;建立了 PCV2和CSFV共感染模型,确定共感染过程中PCV2介导的细胞凋亡是削弱CSFV复制的原因;通过基于iTRAQ的蛋白质组学和生物信息学分析,证明了 PCV2和CSFV共感染过程中PCV2的主导作用,并且确定了共感染过程中重要的蛋白分子及其所涉及的信号通路。然而,这些蛋白和信号通路在共感染过程中的确切功能,还需要进一步研究。
[Abstract]:With the continuous progress and development of large-scale aquaculture technology, the rapid transportation and other factors have increased the chance of contact between the pigs, and also created the opportunity for the epidemic and spread of the pathogens, resulting in the present situation of the mixed infection of two or more pathogens. More and more studies have shown that PCV2 and CSFV can be found in pigs. Co infection, and clinical studies have shown that PCV2 can interfere with the protective efficacy of CSFV attenuated vaccine and bring significant economic losses to the pig industry. However, the interaction between PCV2-CSFV co infection and the mechanism of the impact on the host are unknown. In this study, the polyclonal antibody to the higher titer of the CSFV C strain was obtained by PEG6000 enrichment and sugarcane. The purified CSFV samples were prepared by the sugar density gradient centrifugation method. The results showed that the purified CSFV still had high infection activity and the toxic value was more than 101.5 times of the virus original solution. The purified CSFV was inoculated to New Zealand white rabbit, and the anti CSFV rabbit polyanti sera with high specificity and high titer were obtained. In order to study P effectively In the process of CO infection of CV2 and CSFV and the potential mechanism of cell involvement in virus replication, a model of CO infection in vitro has been established. Under this model, PCV2 is not affected by CSFV replication, and CSFV has no immune rejection to PCV2; while CSFV can reduce.PCV2 and CSFV in the same manner as PCV2 dose dependent manner. CSFV E2 is located in the normal cytoplasm and translocation into the nucleus. In addition, the same dose of PCV2 infection PK15 and PK15-CSFV, PCV2 infection rate is the same, and there is no significant difference in the degree of apoptosis. However, the PCV2 dose and apoptosis show a positive correlation, indicating that PCV2 mediated apoptosis may be down regulated by CSFV replication In addition, inactivation of PCV2 and PCV2 virus component incubating cells can not mediate apoptosis or affect CSFV replication, indicating that PCV2 replication mediated apoptosis affects CSFV replication. These results are sufficient to explain the clinical symptoms of PCV2 and CSFV co infection, as well as the immune failure of the CSFV attenuated vaccine. In order to explore PCV2 and CSFV. The host factors and molecular mechanisms involved in the co infection process, using iTRAQ markers combined with LC-MS/MS mass spectrometry, analyzed the overall proteome of the negative (NE), PCV2 alone infection (SP), CSFV alone infection (SC) and PCV2-CSFV co infection (PC) PK15 samples. In the 3 repeated experiments, a total of 3932 proteins were detected. Ratio of 1.2 or less to 0.83 and P 50.05 as the threshold of down regulation. Compared with NE, the number of down regulated proteins was 304 and 198 respectively, 60 and 61 in SC respectively, and 196 and 156 in PC respectively. The results of relative quantitative PCR, protein immunoblot and confocal laser scanning microscopy proved that most of the genes / proteins were found. The expression trend was consistent with the results of iTRAQ proteome. Clustering, GO and KEGG analysis showed that PCV2 had the leading role in the process of PCV2 and CSFV co infection. The IPA analysis of PC/SC differentially expressed proteins showed that the protein 14-3-3 zeta, cullin3, ERK1/2, caspase and nuclear kappa might have the weight in the process of completing the life cycle in vitro. In addition, the KEGG and IPA analysis of the 184 proteins expressed in the co infection condition were further analyzed. The results showed that the multiple proteins of the mitochondrial respiratory chain complex, I, IV and V were down regulated, and that mitochondria might be the main target of PCV2 and CSFV co infection, and the co infection was also special except for mitochondrial dysfunction. The heterosexual effects on oxidative phosphorylation, Nrf2 mediated oxidative stress, tRNA loading, Myc mediated apoptosis signaling and signaling pathways in various intracellular pathways. These data provide a reference for the study of the effects of PCV2 and CSFV co infection on cell pathways and their molecular mechanisms. In this study, a highly efficient and highly purified CSFV C has been obtained. Strain and anti CSFV rabbit polyacrna provide a tool for detecting CSFV, and a co infection model of PCV2 and CSFV is established to determine that PCV2 mediated apoptosis is the cause of CSFV replication in the process of CO infection. By proteomics and bioinformatics analysis based on iTRAQ, the leading role of PCV2 in PCV2 and CSFV co infection is demonstrated. The important protein molecules and the signal pathways involved in the process of CO infection are determined. However, the exact function of these proteins and signaling pathways in the co infection process needs further study.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651
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本文编号：1775888