本文選題：豬圓環(huán)病毒2型 + 豬瘟病毒�。� 參考：《浙江大學》2016年博士論文

【摘要】：隨著規(guī)�；B(yǎng)殖技術(shù)的不斷進步和發(fā)展、交通運輸?shù)目旖莸纫蛩?增加了豬群之間的接觸機會,也為病原體的流行和傳播創(chuàng)造了機會,造成發(fā)病豬群呈現(xiàn)兩種或多種病原的混合感染的現(xiàn)狀。越來越多的研究表明PCV2和CSFV在豬體內(nèi)能夠發(fā)生共感染,并且臨床的研究表明PCV2能干擾CSFV弱毒疫苗的保護效力,給養(yǎng)豬業(yè)帶來重大的經(jīng)濟損失。然而,PCV2-CSFV共感染時相互作用和對宿主的影響機制還未知。在本研究中,為獲得效價較高的CSFV C株的多克隆抗體,利用PEG6000濃縮法和蔗糖密度梯度離心純化法,制備純化的CSFV樣品。結(jié)果表明,經(jīng)濃縮純化的CSFV仍具有較高的感染活性,且毒價為病毒原液的101.5倍以上。將純化的CSFV作為抗原接種新西蘭大白兔,獲得特異性和效價均較高的抗CSFV兔多抗血清。為了有效地研究PCV2和CSFV共感染過程中病毒的交互影響以及細胞參與病毒復制的潛在機制,建立了一個的體外共感染模型。在該模型下PCV2不受CSFV復制影響,CSFV對PCV2沒有免疫排斥現(xiàn)象;而CSFV的增殖卻以PCV2劑量依賴性的方式降低。PCV2和CSFV能定位于同一個細胞,且CSFV的E2從正常的細胞質(zhì)定位,易位到細胞核中。此外,相同劑量的PCV2感染PK15和PK15-CSFV,PCV2的感染率相同,并且介導的細胞凋亡程度無顯著性差異;然而,PCV2劑量與細胞凋亡呈現(xiàn)正相關(guān),表明PCV2介導的凋亡可能與CSFV復制下調(diào)有關(guān)。另外,滅活PCV2及PCV2病毒成分孵育細胞不能介導細胞凋亡或影響CSFV復制,表明PCV2復制介導的凋亡影響CSFV復制。這些結(jié)果足以解釋臨床上PCV2和CSFV共同感染導致更嚴重的臨床癥狀,以及CSFV弱毒疫苗免疫失敗問題。為了探究在PCV2和CSFV共感染過程中涉及的宿主因子和分子機制,使用iTRAQ標記結(jié)合LC-MS/MS質(zhì)譜鑒定技術(shù),分析陰性(NE)、PCV2單獨感染(SP)、CSFV單獨感染(SC)和PCV2-CSFV共感染(PC)的PK15樣品的整體蛋白質(zhì)組表達情況。在3次重復實驗中,共有3932個蛋白均檢測到。以表達比例≥ 1.2或≤0.83且p 5 0.05作為上下調(diào)的閾值,與NE相比,SP中上、下調(diào)蛋白數(shù)分別為304個和198個,SC中分別為60個和61個,PC中分別為196個和156個。相對定量PCR、蛋白免疫印跡和共聚焦激光掃描顯微鏡技術(shù)驗證的結(jié)果表明,大部分基因/蛋白的表達趨勢與iTRAQ蛋白質(zhì)組結(jié)果一致。聚類、GO和KEGG分析證明在PCV2和CSFV共感染過程中PCV2具有主導作用。對PC/SC差異表達蛋白進行IPA分析,結(jié)果顯示蛋白14-3-3ζ、cullin3、ERK1/2、caspase和NF-κB可能在PCV2影響CSFV在體外完成生命周期的過程中具有重要作用。另外,進一步對共感染條件下特異性差異表達的184個蛋白進行KEGG和IPA分析,結(jié)果表明線粒體呼吸鏈復合物I、IV和V的多個蛋白均下調(diào)表達,由此推斷線粒體可能是PCV2和CSFV共感染時的主要靶標;除了線粒體功能失常外,共感染還特異性影響了氧化磷酸化、Nrf2介導的氧化應激、tRNA裝載、Myc介導的凋亡信號傳導和細胞內(nèi)多種代謝通路的信號通路。這些數(shù)據(jù)為研究PCV2和CSFV共感染過程對細胞通路的影響及其分子機制提供參考。綜上,本研究獲得了高效價高純度的CSFV C株病毒以及抗CSFV的兔多抗,為檢測CSFV提供工具;建立了 PCV2和CSFV共感染模型,確定共感染過程中PCV2介導的細胞凋亡是削弱CSFV復制的原因;通過基于iTRAQ的蛋白質(zhì)組學和生物信息學分析,證明了 PCV2和CSFV共感染過程中PCV2的主導作用,并且確定了共感染過程中重要的蛋白分子及其所涉及的信號通路。然而,這些蛋白和信號通路在共感染過程中的確切功能,還需要進一步研究。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.651
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本文編號：1775888