【作者】 孙英杰；

【导师】 丁铲；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 硕士

【摘要】 新城疫病毒(Newcastle disease virus, NDV)是副黏病毒科、禽腮腺炎病毒属唯一成员,可以感染200余种禽类和哺乳动物,一直是危害世界养禽业的最主要病患之一。根据系统发育进化树分析,可将不同基因组长度的NDV病毒分为两个群,即ClassⅠ(15198nt)和ClassⅡ(15192nt或15186nt)。其中,Class I毒株主要感染水禽和鸟类,这类毒株在该疫病的流行病学和致病机制研究中的重要性一直被人们忽视。本课题组在早期研究中发现一株水禽源NDV Class I毒株,经鸡体传代后,由无毒株(avirulent)演化为强毒株(velogenic),其基因组长度仍为15198nt。为了进一步研究该水禽源NDV对鸡的致病机制和进化适应机理,本文利用该ClassⅠ新城疫病毒强毒株定量感染鸡0系成纤维细胞系(DF-1),通过对感染后各时间点病毒滴度、结构蛋白定位、结构蛋白mRNA水平、细胞骨架和蛋白组学的变化,综合评价ClassⅠ新城疫病毒感染鸡体细胞后的时相情况,对系统地掌握NDV在鸡体细胞中的增殖过程和感染机制具有重要的理论意义。一、Class I新城疫病毒各结构蛋白抗体制备本研究以新城疫病毒(NDV)ClassⅠ强毒离株9a5b尿囊液以及原核表达的NP、P、M、HN、F五种重组蛋白为免疫原,接种6周龄BALB/c小鼠制备抗体,用血凝抑制试验(HI)、间接免疫荧光试验(IFA)、细胞中和试验、酶联免疫吸附试验(ELISA)和Western-blot方法共同检测所获得的抗体。结果显示,成功制备获得了NDV各结构蛋白多抗及12株NDV特异性单抗。免疫特性鉴定结果表明,在获得的12株单抗中,3株为抗ClassⅠNDV特异性单抗(2A8、3H7和3H9),其中,3H7和3H9为HN特异性单抗,仅具有IFA和HI效价;其他9株单抗均与ClassⅡ毒株存在交叉反应。二、ClassⅠN DV强毒株9a5b感染DF1后时相变化研究本研究将9a5b毒株按5m.o.i量感染DF-1细胞,分别于不同时间点收获细胞以及培养上清,利用荧光定量RT-PCR、Western-blot、IFA等方法对病毒结构蛋白mRNA转录水平、蛋白含量及蛋白定位情况进行系统研究。荧光定量RT-PCR结果显示:各结构蛋白mRNA变化与该病毒的生长曲线基本一致;Western-blot和IFA检测结果显示:9a5b病毒感染DF-1后1h就能在细胞浆中检测出NP、P蛋白的存在,其余4种主要蛋白(M、F、HN、L)出现的时间相对较晚,所有病毒蛋白在不同的时间点有着各自特定的定位方式;通过对细胞actin和内质网的染色发现:9a5b病毒感染对这两者的影响并不显著。三、ClassⅠNDV感染CEF蛋白组学初步研究本研究将9a5b、1a毒株按0.001M.O.I.量感染CEF细胞,于感染后12h收获细胞,进行差异蛋白组学研究,结果显示:对1a感染组与CEF对照组利用ImageMasterTM对2-D图谱比较后经质谱分析共检测到差异蛋白点41个其中上调蛋白点13个,下调蛋白点28个,选取其中的11个点进行初步鉴定,成功鉴定出7种蛋白质,对差异蛋白中有重要意义蛋白质的功能进行初步探讨。本研究首次对CLASSⅠNDV感染禽成纤维细胞的时相变化和蛋白组学进行研究,系统的阐述了NDV感染细胞后病毒增殖过程。这为深入开展各蛋白与该病毒的致病机制、毒力演化规律及其与宿主相互作用和进化适应性机制的研究工作奠定了基础。更多还原

【Abstract】 Newcastle disease virus (NDV) is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. It is the causative agent of Newcastle disease (ND), which is widely distributed in more than 200 types of birds and mammals and has inflicted substantial economic costs to poultry industry worldwide. Among the numerous strains, it can be divided into ClassⅠ(15198nt) and ClassⅡ(15192nt or 15186nt) by phylogentic analysis. Because classⅠNDV strains were avirulent and mainly isolated from waterfowls and birds, the epidemiology and pathogenicity of class I NDV in chickens were easy to be neglected. However, in our earlier research, we found a velogenic NDV was generated from a nonpathogenic waterfowl isolate by passaging in chickens. By sequencing the passages, the genomic length maintained with the size of 15198nt.In current study, to research the pathogenicity and evolution mechanism of this waterfowl origin NDV in chickens, Chicken Embryo Fibroblast cell ine (DF-1) was chosed to propagate the velogentic isolate. To systematically know the propagation process and pathogenic mechanism of class I NDV infecting chicken cells, the monolayer of DF1 cell was quantitatively inoculated with classⅠNDV velogenic strain. Cells and cultural supernatant were collected at intervals post infection. Virus titre was evaluated by determination of TCID50, localization of structure proteins in DF1 cells was identified by indirect immunofluorescence assay (IFA), mRNAs level of interest genes was quantitated by real-time RT-PCR and conformation change of cytoskeleton was stained by special dyes.1 Preparation of antibodies against structure proteins of NDVIn the study, several mice were immuned with the virus strain (ClassⅠNDV virulent strain 9a5b) or recombinant proteins expressed in E. coli BL21 (DE3), including Nucleoprotein (NP), Phosphoprotein (P), Matirx (M), Hemagglutinin-neuraminidase (HN), and Fusion (F) proteins. Following by three immunation, the sera were collected. Monoclonal antibodies against NDV were developed by the hybridoma technology. And then the antibodies were identified by hemagglutination inhibition (HI), indirect immunofluorescence assay (IFA), cell neutralization test, ELISA and western-blot analysis. The results showed that, besides anti-sera against each protein, a panel of 12 hybridoma cell lines was successfully prepared. Among them, mAbs 2A8, 3H7 and 3H9 were specific to Class I NDV HN protein in the experiment. All of the other mAbs specific to P or NP protein had a cross-reaction with ClassⅡNDV strains.2 Dynamic properties of interaction of ClassⅠNDV with DF-1 cellsDF-1 cells were inoculated with 9a5b strains at a dose of 5 m.o.i, the cells and cultural supernatant were collected at intervals post infection. Virus titre was evaluated by determination of TCID50, localization of structure proteins in DF1 cells was identified by indirect immunofluorescence assay (IFA), mRNAs level of interest genes was quantitated by real-time RT-PCR and conformation change of cytoskeleton was stained by special dyes. The result shows that, the accumulation of mRNAs were in accordance with the growth curve of the virus. NP and P proteins could be observed in cytoplasm early at 1h post infection, while other proteins were accumulated later relatively. Otherwise, we found that the virus had little effect upon the cytoskeleton by stained of cell actin and Endoplasmic Reticulum with special dyes.3 Preliminary study on proteomics of avian cells infected with ClassⅠNDV.DF-1 cells were inoculated with 9a5b and 1a strains at a dose of 0.001 m.o.i, and then the cells were collected at 12h post infection for proteomics research. Comparison of 2-DE image between 1a infection team and CEF control team revealed that 41 differentially expression protein spots, including 13 up-regulated protein spots, 28 down-regulated protein spots. The 11 protein spots were analyzed by matrixassociated laser dissociation /ionization time of flightmass spectrometry (MALDI-TOF-MS). 7 proteins were successfully identified, among which the protein spots with great importance were preliminary studied.In conclusion, the dynamic properties and proteomics of interaction of ClassⅠNDV with chicken cells were firstly investigated. The propagation mechanism of NDV infection with chicken cells was systematically explained, which is benefit for further study on the mechanism of the virulence evolution and interaction of the class I NDV with avian cells.更多还原