严重急性呼吸道综合征（SARS）S蛋白重组腺病毒疫苗的构建及其免疫学研究

发布时间：2019-06-04 22:31

【摘要】：严重急性呼吸综合征(severe acute respiratory syndrome,SARS),是一种新出现的传染性疾病。由于本病传播速度快,人群普遍易感和死亡率较高,迄今还没有有效的药物和疫苗,目前仍未消除SARS会对人类的健康构成的严重威胁,因此加强SARS防治的基础和应用研究对保障人类的健康和生命安全具有重要意义。 SARS的病原体是一种新的冠状病毒,命名为SARS冠状病毒(SARS Coronavirus SARS CoV)。该病毒属正链RNA病毒,基因组全长约为30kb,与经典冠状病毒仅有约60%同源性,但基因组的组织形式与其它冠状病毒相似。其基因组5′端约三分之二的区域是编码病毒RNA聚合酶复合蛋白,而后三分之一的区域为编码病毒结构蛋白,按基因组上的排列顺序依次为刺突蛋白(S)、表面蛋白(E)、膜蛋白(M)和核蛋白(N)。 S蛋白插在病毒的胞膜上,构成了病毒表面的主要暴露蛋白,形成突起,突起排列间隔较宽。S蛋白为非共价键结合二聚体,是Ⅰ型膜糖蛋白,也是结构蛋白中最大的蛋白,形态学上为鼓锤状。S蛋白有4个结构域:一个短的C末端构成胞质区;一个跨膜区和两个胞膜外域(分别称为S1和S2)。S蛋白具有多方面的功能,负责结合敏感细胞受体、诱导病毒胞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应答。目前,病毒免疫学研究比较公认的观点是,保护性抗体可以中和病毒,使病毒从体内得到清除。SARS-CoV在SARS自愈患者的血清中生长缓慢,说明人体可以产生针对病毒的抗体,这为研制有效的SARS疫苗提供依据。S蛋白是冠状病毒中最主要的抗原,此蛋白在病毒的入侵、中和抗体的产生方面起着非常关键的作用。对最近报道的多株SARS-CoV S蛋白突变位点的分析表明,该蛋白序列上有3个点突变即G77D,1224T,S577A位于S1区段。S2基因较为保守,位于M Helix附近,含有3个呈均匀分布的N连接糖基化位点,可能在S蛋白的免疫原性中起重要作用。因此,本研究选择了SARS-CoV(香港株)的全长S蛋白及其亚单位S1和S2作为SARS疫苗的候选抗原。突起蛋白含有丰富的半胱氨酸,形成15对二硫键。研究表明针对S蛋白的中和性抗体多数是识别构象表位。因此,采用真核分泌表达有利于制备接近天然
[Abstract]:Severe acute respiratory syndrome (severe acute respiratory syndrome,SARS) is a new infectious disease. Due to the rapid spread of the disease and the high prevalence and mortality of the population, there are no effective drugs and vaccines to date, and the serious threat posed by SARS to human health has not yet been eliminated. Therefore, it is of great significance to strengthen the basic and applied research of SARS prevention and treatment to ensure human health and life safety. The pathogen of SARS is a new coronavirus, named SARS coronavirus (SARS Coronavirus SARS CoV). The virus belongs to positive chain RNA virus, and its genome length is about 30kb. it has only about 60% homology with classical coronavirus, but the tissue form of genome is similar to that of other coronavirus. About 2/3 of the 5 'end of the genome encodes the viral RNA polymerase complex protein, and then 1/3 encodes the viral structural protein, followed by the spiny protein (S), surface protein (E), in the order of the genome. Membrane protein (M) and nuclear protein (N). S protein is inserted on the cell membrane of the virus, forming the main exposed protein on the surface of the virus, forming protrusions, and the process arrangement interval is wide. S protein is a non-covalent bond binding dimer, is a type I membrane glycoprotein, and is also the largest protein in the structural protein. Morphologically, the S protein has four domains: a short C-terminal constitutes the cytoplasmic region; One transmembrane region and two outer domains of cell membrane (called S1 and S2, respectively). S protein has many functions and is responsible for binding sensitive cell receptors to induce fusion between viral cell membrane and cell membrane, as well as between cells. Stimulate the production of neutralizing antibodies and mediate cellular immune responses. At present, it is recognized in the study of viral immunology that protective antibodies can neutralize the virus and remove the virus from the body. SARS-CoV grows slowly in the serum of patients with SARS self-healing, indicating that the human body can produce antibodies against the virus. This provides a basis for the development of an effective SARS vaccine. S protein is the most important antigen in coronavirus, which plays a very key role in virus invasion and neutralizing antibody production. The analysis of the recently reported mutation sites of SARS-CoV S protein showed that there were three point mutations in the protein sequence, namely G77D, 1224T, S577A located in S1 region. S2 gene was conserved and located near M Helix. There are three uniformly distributed N-linked glycosylation sites, which may play an important role in the immunogenicity of S protein. Therefore, the full-length S protein and its subunits S1 and S2 of SARS-CoV (Hong Kong strain) were selected as candidate antigens for SARS vaccine. The protruding protein is rich in cysteine and forms 15 pairs of disulfide bonds. Studies have shown that most of the neutralizing antibodies against S protein recognize conformational epitopes. Therefore, the use of eukaryotic secretory expression is beneficial to the preparation of close to natural.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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