SARS-CoV Spike蛋白受体结合结构域在哺乳动物细胞中的表达、纯化及功能研究

【作者】 王顺心；

【导师】 蒋澄宇；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是一种新出现的感染性疾病,其致病体为SARS-CoV。SARS能够导致严重急性肺损伤,许多病人恶化为急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS),具有很高的致死率。此外,受感染者的多个组织、器官受损。SARS具有较高的发病率、致死率,且传播速度快,严重影响了社会、经济的发展。研究表明,Spike(S)蛋白是SARS-CoV表面最重要的结构蛋白,能够介导病毒与宿主细胞受体的结合和膜融合。同时,它能够诱导宿主的免疫反应,产生中和抗体。因此,SARS-CoV S蛋白对于确定SARS-CoV的致病机理及疫苗研发非常重要。由于SARS-CoV的S蛋白本身的编码序列在哺乳动物细胞中的表达量极低,难以满足研究需要。为了解决这个问题,SARS-CoV的S蛋白本身的编码序列被替换为人类基因高频使用的密码子,S蛋白得到高量表达,获得了大量有生物活性的S蛋白。为SARS-CoV S蛋白介导的致病机理的进一步研究和SARS-CoV疫苗的研发提供了基础。SARS-CoV的受体结合结构域(receptor binding domain,RBD)定位于S蛋白的第319到510位氨基酸(AA319-510)。这段多肽能够介导病毒与其受体ACE2结合,决定了病毒的宿主范围和细胞向性。同时,它含有SARS-CoV主要的中和表位,能够诱导中和抗体。通过人类密码子优化的策略,我们获得了大量有生物活性的RBD S蛋白。SARS-CoV进入细胞由S蛋白介导。我们的研究表明,RBD S蛋白能单独通过病毒受体ACE2进入靶细胞。同时,RBD S蛋白N连接的糖基化的去除不能消除RBD S蛋白的这种功能。这些研究将为SARS-CoV感染的分子机制提供新的线索,同时对于靶向病毒进入的治疗性药物的发展提供重要的启示。本论文的最后一部分在哺乳动物细胞中表达了肌肉发育重要抑制因子myostatin。更多还原

【Abstract】 Severe acute respiratory syndrome (SARS) is a new infectious disease caused by SARS coronavirus (SARS-CoV). The clinical presentation indicates that acute lung injury is the major clinical characteristic of SARS and leads to acute respiratory distress syndrome (ARDS) in some severe SARS patients, whose motality is very high. SARS can induce a system disease and impair many other organs apart from lung. Due to its high morbidity and mortality and widespread occurrence, SARS has profoundly disturbed social and economic development.SARS-CoV Spike (S) protein is the most important structural protein on the surface of the SARS-CoV, which forms morphologically characteristic projections on the virion surface, mediates binding to host receptors and membrane fusion. S protein is known to be responsible for inducing host immune responses and virus neutralization by antibodies. S protein is important for pathogenesis study and vaccine development of SARS-CoV. In mammalian cells, the expression level of SARS-CoV S protein encoded by native codons is very low, which is hard to meet the needs of research. To solve this problem, the native codons of SARS-CoV S protein were replaced by codons used frequently by human genes. A large amount of bioactive SARS-CoV S protein was obtained. It is a basement for the research on pathogenesis mediated by SARS-CoV S protein and the development of SARS-CoV vaccine.The receptor-binding domain (RBD) of SARS-CoV is mapped to the amino acids 319-510 on S protein, which can mediate the binding of virus and the virus receptor ACE2, and therefore determine the range of host and cell tropism. At the same time, it contains major neutralization epitopes and can induce neutralization antibody. By human codon-optimization, a large amount of bioactive RBD S protein was obtained. Cell entry of SARS-CoV is mediated by the viral S protein. We demonstrated that RBD S protein alone could enter SARS-CoV susceptible cells through the virus receptor ACE2. We also showed the removal of N-glycans of RBD S protein did not abolish this function. Our discoveries that the RBD S protein alone can enter the cells and the glycosylation of RBD spike protein has no effect on the virus entry provide additional elucidation on the molecular mechanism of SARS-CoV infection. These might be important implications for the development of therapeutic drugs targeted to virus entry.The last part of this thesis is the expression of an important inhibitor to muscle development, myostatin, in mammalian cells.更多还原