本文選題：CPV + CPV-2c　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：犬細(xì)小病毒(Canine Parvovirus,CPV)是最常見的犬的致死性病毒之一。CPV在世界范圍內(nèi)流行,變異率很高,但是該病沒有有效治療方法,主要通過(guò)疫苗免疫進(jìn)行預(yù)防。為了監(jiān)測(cè)我國(guó)東北部地區(qū)的CPV變異情況,更好的預(yù)防CPV感染,從哈爾濱、長(zhǎng)春、沈陽(yáng)、保定、青島收集216份臨床病料,測(cè)序獲得64株VP2全長(zhǎng)序列,包含40株CPV-2a型,23株CPV-2b型和1株CPV-2c型毒株。調(diào)查結(jié)果顯示,保定地區(qū)主要流行CPV-2b型毒株,其它4個(gè)地區(qū)主要流行CPV-2a型毒株,CPV-2c型首次在青島地區(qū)得到檢測(cè)。突變位點(diǎn)分析表明,本實(shí)驗(yàn)中100%的毒株序列在297和324位氨基酸殘基分別發(fā)生Ser-Ala和Ile-Arg的突變。我們檢測(cè)到5株病毒存在Gln370Arg突變,以往這種突變僅在熊貓的CPV分離株中發(fā)現(xiàn),該突變可能會(huì)影響CPV的宿主范圍。選擇壓力預(yù)測(cè)發(fā)現(xiàn)突變Phe267Tyr,Ser297Ala,Thr324Ile和Thr440Ala均在正向選擇壓力下。ML進(jìn)化樹顯示,中國(guó)與其他國(guó)家流行的CPV-2c型毒株存在顯著地域差異。MCC樹預(yù)測(cè)發(fā)現(xiàn),東北部地區(qū)當(dāng)前流行的64個(gè)毒株都在同一進(jìn)化分支上,它們最近的共同祖先出現(xiàn)在1996年。值得注意的是,這次流行病學(xué)調(diào)查發(fā)現(xiàn)以CPV-HLJ-6為代表18株CPV-2a序列完全一致,表明該毒株為東北部地區(qū)的優(yōu)勢(shì)流行毒株,我們推測(cè)當(dāng)前使用的疫苗不能對(duì)該優(yōu)勢(shì)毒株產(chǎn)生足夠的免疫效力。為了揭示CPV流行毒株與疫苗毒株之間的差異,我們利用iTRAQ結(jié)合納米高效液相色譜串聯(lián)質(zhì)譜技術(shù),在優(yōu)勢(shì)流行毒株CPV-HLJ-6和CPV-2型弱毒疫苗毒株感染F81細(xì)胞后48 h進(jìn)行比較蛋白質(zhì)組學(xué)分析。鑒定到31個(gè)差異表達(dá)蛋白,利用David和Panther功能富集分析發(fā)現(xiàn),差異蛋白涉及31個(gè)生物學(xué)過(guò)程,富集于14個(gè)細(xì)胞組分,分類于9個(gè)分子功能。利用MetaCore進(jìn)行通路富集分析發(fā)現(xiàn),差異蛋白顯著富集于血凝、細(xì)胞骨架重塑和G蛋白信號(hào)相關(guān)信號(hào)通路。Ras超家族蛋白質(zhì)、細(xì)胞骨架相關(guān)蛋白質(zhì)以及血凝現(xiàn)象相關(guān)蛋白的發(fā)現(xiàn),有助于從宿主細(xì)胞對(duì)病毒復(fù)制的影響、細(xì)胞致病性、病毒血凝性等方面揭示流行毒株與疫苗毒株之間的差異。CPV流行毒株與疫苗毒株感染細(xì)胞的蛋白種類及表達(dá)量差異可能是毒株不同導(dǎo)致的,也可能是細(xì)胞處于不同感染階段所致。為了在感染后各個(gè)階段解析CPV與宿主相互作用關(guān)系,我們對(duì)CPV感染F81細(xì)胞后的5個(gè)時(shí)間點(diǎn)進(jìn)行了比較蛋白質(zhì)組學(xué)分析。通過(guò)方差分析,篩選到感染后0-60 h存在615個(gè)差異蛋白質(zhì),通過(guò)雙尾未配對(duì)t檢驗(yàn),分別篩選到85、102、95、209和225個(gè)細(xì)胞蛋白質(zhì)的表達(dá)量在CPV感染12 h、24 h、36 h、48 h和60 h有顯著差異。韋恩分析發(fā)現(xiàn)僅有3個(gè)差異蛋白質(zhì)共同存在于5個(gè)時(shí)間點(diǎn)。David功能富集發(fā)現(xiàn),感染后12 h的差異蛋白主要富集于細(xì)胞周期相關(guān)生物學(xué)過(guò)程,在感染后60 h細(xì)胞凋亡相關(guān)差異蛋白種類最多。結(jié)合David及Cluster3.0分析,獲得了大量與細(xì)胞凋亡和病毒刺激相關(guān)的蛋白質(zhì)。通過(guò)通路富集分析發(fā)現(xiàn)8個(gè)與CFTR降解通路相關(guān)的蛋白質(zhì),可能與CPV感染致病性密切相關(guān)。最后,利用Western blot方法,分別驗(yàn)證了7個(gè)蛋白質(zhì)在5個(gè)不同時(shí)間點(diǎn)的表達(dá)情況。結(jié)合以上結(jié)果,我們?cè)诓町惖鞍字邪l(fā)現(xiàn)了與細(xì)胞有絲分裂、細(xì)胞凋亡、病毒感染刺激及病毒生命活動(dòng)相關(guān)的蛋白質(zhì),為研究CPV復(fù)制、凋亡、致病機(jī)制及宿主抗病毒機(jī)制提供了新思路。該實(shí)驗(yàn)填補(bǔ)了我國(guó)東北部CPV流行病學(xué)及CPV蛋白質(zhì)組學(xué)研究的空白,為CPV疾病的防控提供了理論基礎(chǔ),為該病毒蛋白質(zhì)組學(xué)研究搭建了平臺(tái)。
[Abstract]:Canine Parvovirus (CPV) is one of the most common fatal viruses in dogs..CPV is popular in the world and has a high rate of variation. However, there is no effective treatment for this disease, but it is mainly prevented by immunization. In order to monitor the variation of CPV in the northeast of China, it is better to prevent CPV infection, from Harbin, Changchun, Shen. 216 clinical materials were collected from Yang, Baoding and Qingdao, and 64 VP2 full-length sequences were sequenced, including 40 CPV-2 a, 23 CPV-2b and 1 CPV-2c strains. The results showed that the main epidemic CPV-2b strains in Baoding area, the other 4 areas were mainly CPV-2a type strains, and the CPV-2c type was first detected in Qingdao area. Mutation site analysis was analyzed. In this experiment, 100% of the strains were mutated with Ser-Ala and Ile-Arg in the 297 and 324 amino acid residues, respectively. We detected that 5 strains of virus had Gln370Arg mutations. The mutation was found only in the CPV isolate of the panda, and the mutation may affect the host enclosure of CPV. La, Thr324Ile and Thr440Ala were all.ML evolutionary trees under positive selective pressure. There was a significant regional difference between China and other popular CPV-2c strains,.MCC tree prediction found that 64 strains of current prevalent strains in the northeastern region were in the same evolutionary branch, and their recent common ancestor appeared in 1996. The epidemiological survey found that the 18 CPV-2a sequences represented by CPV-HLJ-6 were identical, indicating that the strain was the dominant strain in the northeastern region. We speculated that the current vaccine could not produce enough immune effect to the dominant strain. In order to reveal the difference between the CPV strains and the vaccine strains, we use iTRAQ binding Nana. High performance liquid chromatography tandem mass spectrometry (MS) was used to compare the proteomic analysis of 48 h infected F81 cells with the dominant strain CPV-HLJ-6 and CPV-2 virus strain. The differential protein was identified by David and Panther enrichment analysis. The difference protein was involved in 31 biological processes and enriched in 14 cell components. It was classified into 9 molecular functions. MetaCore pathway enrichment analysis showed that the difference protein was significantly enriched in blood coagulation, cytoskeleton remodeling and G protein signal related signaling pathway.Ras superfamily proteins, cytoskeleton related proteins and the discovery of hemagglutination related proteins, which contributed to the effect of host cells on virus replication. The differences between the virus strains and the vaccine strains, the difference in protein types and expression of infected cells between.CPV epidemic strains and vaccine strains may be caused by different strains and may be caused by different stages of infection. In order to analyze the interaction between CPV and host at various stages after infection. We made a comparative proteomics analysis on the 5 time points after CPV infection of F81 cells. Through the analysis of variance, 615 differential proteins were screened for 0-60 h after infection. Through double tail unpaired t test, the expression of 85102,95209 and 225 cell proteins were screened respectively in CPV infection 12 h, 24 h, 36 h, 48 h and 60 h. There were significant differences. Wayne analysis found that only 3 differential proteins existed at 5 time points with.David enrichment, and the difference proteins of 12 h after infection were mainly enriched in cell cycle related biological processes, and the number of different proteins related to apoptosis in 60 h cells after infection was the most. Proteins associated with apoptosis and viral stimulation. Through pathway enrichment analysis, 8 proteins associated with the CFTR degradation pathway were found to be closely related to the pathogenicity of CPV infection. Finally, the expression of 7 proteins at 5 different time points was verified by Western blot method. Proteins related to cell mitosis, apoptosis, virus infection and viral life activities have been found, which provide new ideas for the study of CPV replication, apoptosis, pathogenicity mechanism and host virus mechanism. This experiment fills the gap of CPV epidemiology and CPV proteomics research in northeastern China, and provides the prevention and control of CPV disease. The theoretical basis provides a platform for the study of viral proteomics.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.292

【參考文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 李丹;血紅蛋白β亞基對(duì)豬瘟病毒增殖的調(diào)控作用及其調(diào)節(jié)機(jī)制[D];中國(guó)農(nóng)業(yè)科學(xué)院;2013年

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