犬细小病毒流行病学调查及其感染F81细胞的比较蛋白质组学分析

发布时间：2018-06-02 00:28

本文选题：CPV + CPV-2c　；参考：《中国农业科学院》2016年博士论文

【摘要】：犬细小病毒(Canine Parvovirus,CPV)是最常见的犬的致死性病毒之一。CPV在世界范围内流行,变异率很高,但是该病没有有效治疗方法,主要通过疫苗免疫进行预防。为了监测我国东北部地区的CPV变异情况,更好的预防CPV感染,从哈尔滨、长春、沈阳、保定、青岛收集216份临床病料,测序获得64株VP2全长序列,包含40株CPV-2a型,23株CPV-2b型和1株CPV-2c型毒株。调查结果显示,保定地区主要流行CPV-2b型毒株,其它4个地区主要流行CPV-2a型毒株,CPV-2c型首次在青岛地区得到检测。突变位点分析表明,本实验中100%的毒株序列在297和324位氨基酸残基分别发生Ser-Ala和Ile-Arg的突变。我们检测到5株病毒存在Gln370Arg突变,以往这种突变仅在熊猫的CPV分离株中发现,该突变可能会影响CPV的宿主范围。选择压力预测发现突变Phe267Tyr,Ser297Ala,Thr324Ile和Thr440Ala均在正向选择压力下。ML进化树显示,中国与其他国家流行的CPV-2c型毒株存在显著地域差异。MCC树预测发现,东北部地区当前流行的64个毒株都在同一进化分支上,它们最近的共同祖先出现在1996年。值得注意的是,这次流行病学调查发现以CPV-HLJ-6为代表18株CPV-2a序列完全一致,表明该毒株为东北部地区的优势流行毒株,我们推测当前使用的疫苗不能对该优势毒株产生足够的免疫效力。为了揭示CPV流行毒株与疫苗毒株之间的差异,我们利用iTRAQ结合纳米高效液相色谱串联质谱技术,在优势流行毒株CPV-HLJ-6和CPV-2型弱毒疫苗毒株感染F81细胞后48 h进行比较蛋白质组学分析。鉴定到31个差异表达蛋白,利用David和Panther功能富集分析发现,差异蛋白涉及31个生物学过程,富集于14个细胞组分,分类于9个分子功能。利用MetaCore进行通路富集分析发现,差异蛋白显著富集于血凝、细胞骨架重塑和G蛋白信号相关信号通路。Ras超家族蛋白质、细胞骨架相关蛋白质以及血凝现象相关蛋白的发现,有助于从宿主细胞对病毒复制的影响、细胞致病性、病毒血凝性等方面揭示流行毒株与疫苗毒株之间的差异。CPV流行毒株与疫苗毒株感染细胞的蛋白种类及表达量差异可能是毒株不同导致的,也可能是细胞处于不同感染阶段所致。为了在感染后各个阶段解析CPV与宿主相互作用关系,我们对CPV感染F81细胞后的5个时间点进行了比较蛋白质组学分析。通过方差分析,筛选到感染后0-60 h存在615个差异蛋白质,通过双尾未配对t检验,分别筛选到85、102、95、209和225个细胞蛋白质的表达量在CPV感染12 h、24 h、36 h、48 h和60 h有显著差异。韦恩分析发现仅有3个差异蛋白质共同存在于5个时间点。David功能富集发现,感染后12 h的差异蛋白主要富集于细胞周期相关生物学过程,在感染后60 h细胞凋亡相关差异蛋白种类最多。结合David及Cluster3.0分析,获得了大量与细胞凋亡和病毒刺激相关的蛋白质。通过通路富集分析发现8个与CFTR降解通路相关的蛋白质,可能与CPV感染致病性密切相关。最后,利用Western blot方法,分别验证了7个蛋白质在5个不同时间点的表达情况。结合以上结果,我们在差异蛋白中发现了与细胞有丝分裂、细胞凋亡、病毒感染刺激及病毒生命活动相关的蛋白质,为研究CPV复制、凋亡、致病机制及宿主抗病毒机制提供了新思路。该实验填补了我国东北部CPV流行病学及CPV蛋白质组学研究的空白,为CPV疾病的防控提供了理论基础,为该病毒蛋白质组学研究搭建了平台。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.292

【参考文献】