【作者】 王宝梅；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2004， 博士

【摘要】 严重急性呼吸综合征(severe acute respiratory syndrome，SARS)，亦称传染性非典型肺炎，是一种新出现的急性呼吸道传染病。自2002年11月中旬广东省佛山市发现首宗病例以来，该病迅速蔓延到包括我国香港特别行政区和台湾地区、越南、新加坡、加拿大、德国在内的五大洲的33个国家及地区，至少在6个国家中发生了本地传播。由于此病传播速度快，人群普遍易感而且死亡率较高，已成为严重危害人类健康和生命安全的全球性流行性疾病。该病主要通过近距离飞沫和密切接触传播。主要临床特征为：发热、干咳、呼吸困难、淋巴细胞减少以及肺部影像学呈迅速进行性改变，上呼吸道症状并不显著，部分患者有腹泻症状，一些病人可因严重呼吸窘迫致呼吸衰竭而死亡。使用传统的抗生素治疗无效。在世界卫生组织(WHO)的协调下，全球9个国家13个实验室共同努力，仅用3周的时间就分离出病原体，并完成全基因组测序。2003年4月16日，WHO正式宣布一种以前未曾报道过的新的冠状病毒为导致SARS的病原体，并命名为SARS冠状病毒(SARS Coronavirus，SARS-CoV)。 SARS冠状病毒粒子呈球状，但具有多形性，其直径80～220nm，有包膜，包膜上有3种糖蛋白，分别为S蛋白(spike protein)、M蛋白(membrane protein)和E蛋白(envelope protein)。由S蛋白组成的突起长度为20nm，每个突起是由3个S蛋白通过非共价键连接而成。S蛋白有4个结构域：一个短的C末端构成包质域；一个跨膜区和两个包膜外域(分别称为S1和S2)。包质域富含保守的半胱氨酸残基，与S蛋白形成复杂的三级结构有关。跨膜区与S2蛋白相连，含有一段高度保守的序列KWPWYVWL。通常包质域和跨膜区在S蛋白中所占比例很小，大部分是由S1和S2构成的包膜外域。S蛋白三聚体具有多方面的功能，负责结合敏感细胞受体，诱导病毒包膜和细胞膜以及细胞之间的融合，刺激机体产生中和抗体和介导细胞免疫反应。 细胞毒T淋巴细胞(cytotoxic T lymphocyte，CTL)在机体抵抗各类病毒感博士论文SARS冠状病毒S蛋白HLA一A*0 201限制性COS+T淋巴细胞表位的鉴定染和/或导致免疫病理损伤中起着极为重要的作用。淋巴细胞减少症 (lymPhoPenia)是SARS发病早期一个重要的临床症状。鉴于SARS侵袭了亚洲的许多国家和地区，且HLA，AZ阳性人群在亚洲人中所占比例很高(>50%)，因此，我们以S蛋白为靶标，联合使用CTL表位的远程预测，超基序方案，延展基序法及量化基序法，预测并合成了20条最有可能的HLA一A*0201限制性的CDS+T淋巴细胞候选表位，通过肤结合实验，筛选出与HLA一A*0201具有高亲和力的九肤，并对其免疫原性进行评价，发现SSp一1(SARS/S一derived pePtide一1，RLNEVAKNL)是一个可被细胞自然加工、递呈的免疫原性多肤。树突状细胞(dendritic cen，DC)在抗原肤体内外诱导细胞免疫应答中具有强有力的佐剂效应，以ssp一1体外致敏Dc，分别在HLA一A2.1服b转基因小鼠及健康人外周血淋巴细胞中诱导出了抗原特异性的、HLA一A*0201限制性的细胞毒T淋巴细胞:并且我们还构建了包含ssp一1的HLA一A*0201四聚体(SSp一l/HLA一A*0201 tetramer)，用于检测抗原肤特异性CTL的频率。SARS冠状病毒S蛋白来源的、HLA一A*O加1限制性的CDS+T淋巴细胞表位的鉴定，有助于研究CTL在SARS病毒控制和病理损伤中发挥的作用，对阐明SARS发病机制以及免疫干预治疗的研究均有重要的意义。第一部分SARS一COVS蛋白HLA一A*0 201限制性CDS+T淋巴细胞表位的预测及初步鉴定 随着分子生物学和分子免疫学技术的发展，尤其是计算机科学在生物医学中的广泛应用，目前己产生了多种CTL表位预测方案。我们以SARS病毒BJ01株基因组序列为基础，联合使用CTL表位的远程预测、超基序方案、延展基序法及量化基序法，预测了20个最有可能的HLA·A*0201限制性的CDS+T淋巴细胞候选表位，并通过肤结合实验对其进行了初步筛选。SARS一CoVS蛋白HLA一A*0201限制性CDS+T淋巴细胞表位的预测 通过美国国立卫生研究院生物信息及分子分析部(BIMAs) HLA肤结合预测网站‘http://b imas.dcrt.nih.gov/m olbio小la._bind/inde茸.html)，对SARS一CoV BJ01博士论文SARS冠状病毒S蛋白HLA·A*0 201限制性COS+T淋巴细胞表位的鉴定stl妞in(GenBank aceession number灯278488)的s蛋白进行T分析，选取预测SARS一CoVS抗原中HLA一A*0201/抗原肤复合物解离时间(Tl/2)最长的20个候选九肤，其序列分别为No.1 VVFLHVTYV(1042一1050)、No.2 RLQSLQTYV (982一990)、No，3 VLNDILSRL(958一966)、No.4 VLYENQKQI(897~905)、No.5 NLNESLIDL(1 174一1182)、No.6 LLLQYGSFC(734一742)、No.7KLPDDFMGC(411~419)、No.8 ILPDPLKPT(787一795)、No.9 MIFDNAFNC (151一159)、No.10 FIFLLFLTL(10~18)、No.ll VLYQDVNCT(596一604)、No.12 IITTDNTFV(1096一1 104)、No.13 RLNEVAKNL(1167、1175)、No.14LLTDDMIAA(846一854)、No.15 LLQYGSFCT(735、743)、No.16 ILLCCMTSC(1214一1222)、No.17 LLFNKVTLA(803一811)、No.18 TMNNKSQSV(106一114)、No.19 ALNTLVKQL(940一948)、No.ZO ELCDNPFFA(131一139)。 所预测?更多还原

【Abstract】 The severe acute respiratory syndrome (SARS), also named infectious atypical pneumonia, is a newly described and highly contagious respiratory disease that first occurred in mid-November of 2002 in Guangdong Province, China, and spread to 33 countries around the world. The mortality of the disease is about 11% (range 7 to 27%) for the most severely affected regions as reported by WHO. SARS has been a growing threat to public health. The disease is characterized by high fever, rigor, headache, lymphopenia, nonproductive cough or dyspnea and may progress to generalized, interstitial infiltrates in the lung, requiring intubation and mechanical ventilation. Some cases progress and deteriorate rapidly to acute respiratory distress syndrome (ARDS), multiorgan failure within 2~3 weeks or even in a much shorter time and easily leading to death. To control the spread of SARS, the World Health Organization (WHO) coordinated extraordinary collaborations among laboratories around the world to identify the causative agent of SARS and completely sequence the virus genome. In April 2003, WHO announced that SARS-coronavirus (SARS-CoV), a new member of the coronavirus family was the aetiological agent of SARS.The SARS-CoV, a positive-stranded RNA virus, has four structural proteins, spike (S), envelope (E), membrane (M) and nucleocapsid (N). The S protein, with its size of 1,255 amino acids and 23 glycosylation sites, is the largest structural protein in the SARS-CoV. Its amino acid sequence is significantly different from that of the other known coronaviruses, with only 24% pairwise amino acid identity, while the topology of this protein is similar to that of the other known coronaviruses. The S protein usually has a’ huge amount of ectodomains and short transmembrane spans. The majority of amino acids of the SARS-CoV S protein at the N-terminus (residues 14 to1,195) were found on the outside of the viral particle, while only 13 amino acids at the C-terminus were embedded in the viral membrane. The S protein is responsible for viral attachment to cell receptors and entry into susceptible cells, eliciting neutralizing antibodies and inducing cytotoxic T lymphocyte (CTL) response.A common observation in patients with SARS is pronounced lymphonenia and a notable drop in CD4+ and CD8+ T lymphocyte counts occurring early in the course of the disease is associated with adverse outcomes. Evidence suggests that CD8+ cytotoxic T lymphocytes (CTLs) play a pivotal role in both virus elimination and immunopathology induction in acute respiratory virus infections, following recognition of epitopes presented on target cells in the context of MHC class I. But the molecular mechanisms underlying these CD8+ CTL-mediated effects remain poorly defined. HLA-A2 is the most common HLA-A allele in Asian populations, particularly in Chinese, with an estimated frequency of more than 50%. As SARS has affected many parts of Asia and a reservoir of infection may persist in these regions, the identification of HLA-A*0201 -restricted CTL epitopes of SARS-CoV can contribute greatly to the studies concerning the role of CTLs in SARS-CoV pathogenesis and the protection in at-risk populations.In this study, twenty peptides with binding motifs for HLA-A*0201 were predicted using computer algorithm and verified via MHC-peptide binding assay. Then the capacities of high-affinity candidate peptides to induce CTL in vivo and in vitro were further investigated. We have identified a novel HLA-A* 0201-restricted, immunogenic CD8+T-cell epitope of the SARS-CoV S protein, SSp-1 (RLNEVAK NL). SSp-1 is not only able to induce specific CTL response in HLA-A2.1/Kb transgenic mice but can also mobilize a specific CTL repertoire in PBLs from healthy HLA-A2.1"1" human donors. Moreover, SSp-1 is a naturally processed immunogenic peptide and is recognized by CTLs in an antigen-specific and HLA-A* 0201-restricted manner. Furthermore, we have established a sensitive and specific assay for peptide-specific CTL based on SSp-l/HLA-A*0201 tetramers. To the best of our knowledge,更多还原