The Study On Expression And Purification Of Coronavirus Spike Protein Trimer And It's Binding To Monoclonal Antibody

Coronaviruses(CoVs)are single-stranded,positive-sense RNA viruses with the largest genome and cause mild or lethal respiratory and gastrointestinal diseases in humans and animals.The N-terminus of the coronavirus genome contains two large open reading frames(ORFs),ORF1 a and ORF1b.The C-terminus mainly contains genes encoding four structural proteins:S protein(spike glycoprotein),M protein(membrane protein),E protein(envelope protein)and N protein(nuclear protein).S glycoprotein is a homotrimeric class I fusion protein that consists of three S1 heads and a trimeric S2 stalk.During virus entry,the S1 subunit binds to a receptor on the host cell surface and initiate a series of conformational changes in the spike,the S2 subunit fuses the host and viral membranes,allowing viral genomes to enter host cells.The spike(S)glycoprotein on the coronavirus,which acts as both the major determinant of host cell tropism and the mediator of viral entry into host cells.Thus far,Cryo-EM structure of virus-Fab complexes are available for SARS-CoV?MERS-CoV.Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design.In this study,the expression and purification of PEDV S protein trimer and the binding test with monoclonal antibody were taken as an example,and the S protein trimer of SeACoV and SARS-CoV-2 was expressed and purified in turn.which lays the foundation for the Cryo-EM structure of protein-antibody complex.The specific research is as follows:1 We optimized and reconstructed the S sequence,construct a recombinant plasmid.By using the Bac-to-Bac insect cell system,we expressed PEDV S protein.Then S glycoprotein was purified by affinity and gel filtration chromatography.Cross-linking and density gradient centrifugation were used to optimize protein quality.Fractions containing PEDV S protein were pooled,concentrated then observed by negative-stain EM.2 By SDS-PAGE and Western blot analysis,the PEDV S protein trimer was used to identify four monoclonal antibodies of PEDV S monomer protein existing in the laboratory.3 We expressed the S protein trimer of porcine intestinal alphacoronavirus(SeACoV)and the new coronavirus(SARS-CoV-2)by using the same expression conditions as the PEDV S protein trimer.