Sars-cov Spike Protein Receptor Binding Domain Of Expression In Mammalian Cells, Purification And Functional

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.