【作者】 曹中赞；

【导师】 刘胜旺；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 博士

【摘要】 冠状病毒具有广泛的宿主范围,能够导致人和多种动物出现不同程度的呼吸道、肠道和神经系统的功能紊乱症状。2003年春季SARS-CoV的出现及其感染并导致的高死亡率引起了人们对冠状病毒的高度重视。对冠状病毒的致病性以及致病机理的研究是当前研究者关注的热点之一。IBV属于γ冠状病毒,主要在家禽的气管、肾脏、腺胃和生殖道等器官组织的上皮细胞中复制。IBV的传播给全球家禽业带来严重的经济损失。已有的研究证明部分其他冠状病毒的感染能够影响宿主细胞的蛋白表达模式。本研究首次利用蛋白质组学以及相关的分子生物学技术对人工感染IBV鸡胚的气管和肾脏组织的差异表达蛋白质、人工感染不同毒力IBV后不同阶段的鸡气管和肾脏组织的差异表达蛋白质进行了鉴定,并应用生物信息学工具对差异表达蛋白质的分子功能、生物学进程和亚细胞分布进行分类,旨在从蛋白质水平上探索IBV与宿主的相互作用以及IBV的致病机制。本研究首先利用双向凝胶电泳（2-DE）结合质谱技术对人工感染IBV H120疫苗株72小时时鸡胚和未感染对照组鸡胚气管和肾脏组织的群体蛋白质表达情况进行了分析。鉴定出了一些有显著差异表达的蛋白质。生物信息学分析表明这些蛋白质大多与细胞骨架组成、钙离子结合、能量代谢、抗氧化、应激反应、以及大分子物质合成有关,其中有些蛋白还具有调节细胞凋亡的特性。应用荧光定量PCR技术对热休克蛋白beta-1、胞外脂肪酸结合蛋白、原肌球蛋白alpha-1、膜联蛋白A5、过氧化物还原酶-1、膜联蛋白A2、烯醇酶-1等11种蛋白的转录水平进行了分析,结果表明这些蛋白基因的mRNA水平变化趋势和2-DE结果基本一致。Western blot分析进一步验证了热休克蛋白beta-1和膜联蛋白A5的表达变化,结果与mRNA表达模式的变化也一致。本研究着重应用2-DE和荧光差异显示双向凝胶电泳（2-DIGE）结合质谱技术研究了人工感染IBV强毒株ck/CH/LDL/97I P5和其鸡胚传代致弱毒株ck/CH/LDL/97I P115后不同时间点鸡气管和肾脏组织群体蛋白质的表达改变情况,鉴定出62种差异表达蛋白。生物信息学分析表明大部分差异蛋白与细胞骨架组成、急性期应答、抗氧化、应激应答、能量代谢,大分子物质代谢,信号转导和离子运输有关。用荧光定量PCR技术对热休克蛋白beta-1、膜联蛋白A2、波形蛋白、膜联蛋白A5、锰超氧化物歧化酶、线粒体磷酸烯醇丙酮酸羧基酶等14种差异表达蛋白进行转录水平的分析,其动态变化趋势与蛋白质组学的结果大部分是一致的。用western blot方法对三种差异表达蛋白膜联蛋白A2、膜联蛋白A5和热休克蛋白beta-1从表达水平上进行了验证,其结果与蛋白质组学得到的结果是一致的。实验结果表明,IBV强毒株ck/CH/LDL/97I P₅和鸡胚传代致弱毒株ck/CH/LDL/97I P₁₁₅感染能够诱导包括细胞骨架组成、抗氧化、应激应答、能量代谢等相关蛋白在内的一些在宿主应答过程中可能起重要作用的关键蛋白产生不同模式的表达变化,而且这种表达变化的差异主要发生在感染的早期阶段。本研究首次获得了较为完善的人工感染IBV自然宿主鸡的气管和肾脏组织的差异表达蛋白质数据,为进一步阐释IBV的致病机理,以及筛选到有价值的预防或治疗药物提供了有价值的信息。同时,本研究结果也为SARS-CoV等其他冠状病毒的相关研究提供了参考依据。更多还原

【Abstract】 Coronaviruses can infect a broad range of host including human and animal, cause respiratory, gastrointestinal, and neurological disorder of varying severity. Since the spring of 2003, Coronaviruses were brought to the centre of attention by the appearance of the severe acute respiratory syndrome-associated coronavirus （SARS-CoV） and caused high mortality. Research on the pathogenesis and pathogenicity of coronaviruses is the current focus of attention. Infectious bronchitis virus （IBV） is belonging to gamma coronavirus, which replicates primarily in a large range of epithelial cells of trachea, kidney, proventriculus, and oviduct of chicken, and probably endemic in all regions with intensive impact on poultry production. Previous studies demonstrated that some other coronaviruses infection have dramatic effects on the patterns of host protein expression. In order to understand the IBV-host interaction and the pathogenesis of IBV, in this study, we for the first time used proteomics techniques and other molecular biological methods to study the global protein expression change profiles of trachea and kidney from chicken embryos following IBV H120 vaccine strain infection in ovo, and the trachea and kidney tissues protein expression changes of chickens during different stages after infection in vivo with highly virulent IBV ck/CH/LDL/97I P5 strain and embryo-passaged, attenuated IBV ck/CH/LDL/97I P115 strain. Gene Ontology annotation analysis of differentially expressed proteins was performed according their molecular function, biological process, and subcellular location. The aim of this study is to investigate the interactions of IBV with its host, and the pathogenesis of IBV at the protein level.The changes of protein expression in trachea and kidney of chicken embryo at 72h after infection in ovo with IBV H120 vaccine strain were first analyzed using 2-DE coupled with MALDI-TOF/TOF MS method. Seventeen differentially expressed proteins from tracheal tissues and nineteen differentially expressed proteins from kidney tissues were identified successfully. These proteins mostly related to the cytoskeleton organization, binding of calcium ions, response to stress, anti-oxidative, energy metabolism, and macromolecular biosynthesis. Notably, some of the identified proteins have the ability of regulation apoptosis. Eleven altered proteins including annexin A1, annexin A2, annexin A5, tropomyosin 1 alpha, peroxiredoxin-1, Calbindin-D28k, heat shock protein beta-1, myosin light chain type 2, extracellular fatty acid-binding protein, TRIM27.2, and enolase alpha were analyzed further at the mRNA level using real-time PCR. The trends of the changes in their mRNA levels were similar to the patterns of change in their corresponding proteins on 2-DE gels. Western blot analysis further confirmed the changes of annexin A5 and HSPB1. It was also in consisting with results obtained by real-time PCR.We focus on study the global protein expression changes in trachea and kidney tissues of chickens during different stages after infection in vivo with highly virulent IBV ck/CH/LDL/97I P5 strain and embryo-passaged, attenuated IBV ck/CH/LDL/97I P115 strain using 2-DE and 2-DIGE coupled with MALDI-TOF/TOF MS method. In total, sixty-two differentially expressed proteins were identified and classified into several functional categories including cytoskeletal organization, macromolecule metabolism, anti-oxidative stress, stress response, acute phase response, signal transduction and ion transport. The dynamic transcriptional alterations of fourteen selected proteins including heat shock protein beta-1, annexin A2, annexin A5, tropomyosin alpha, vimentin, lamin A, manganese-containing superoxide dismutase, and phosphoenolpyruvate carboxykinase mitochondrial were analyzed using real-time PCR method. The majority mRNA level changed trends of these genes were consistent with the change patterns of their corresponding proteins in 2-DE and 2-DIGE gels. Western blot analysis further confirmed the expression changes of HSP beta-1, annexin A2, and annexin A5 obtained by proteomics method. Results demonstrated that some key proteins which were likely to be important in the host response to virus infection, including cytoskeleton organization, anti-oxidative stress, stress responses, and energy metabolism, were resulted in different expressed change patterns between infection with the highly virulent IBV ck/CH/LDL/97I P5 strain and its embryo-passaged, attenuated P115 stain. Additionally, these dramatic differences between P₅- and P₁₁₅-strain induced changes were mainly observed at the early stage of virus infection.In general, these results provide valuable insights into the interactions of IBV with its host, the investigations in pathogenesis of IBV, and screening of preventive and therapeutic medicine. Meanwhile, our study may provide a reference for related research of SARS-CoV and other coronaviruses.更多还原