Expression And Purification Of Coronavirus Spike Protein S2 Subunit By Baculovirus Expression System

Coronaviruses are enveloped viruses with a linear positive sense single stranded RNA genome.They have the largest genome among known RNA viruses and are widely found in nature.Coronaviruses are divided into α、β、γ and δ four subgroups.Severe acute respiratory coronavirus 2(SARS-Co V-2)belongs to β Type coronavirus.SARS-Co V-2 has a strong infectivity and pathogenicity in human beings.Meanwhile,SARS Co V-2 has strong mutation ability,and a lot of mutant strains including Alpha,Beta,Gamma,Delta,Omicron strains have been produced within the last three years of the pandemic.The viral genome of coronavirus is wrapped in a lipid bilayer membrane,with spike glycoproteins(S)anchored on the surface.Sprotein on coronavirus particle mediates the virus entry into the target cells.The S1 subunit is responsible for recognizing and binding to the angiotensin converting enzyme 2(ACE2)receptor on the host cell,and the S2 subunit is responsible for the membrane fusion of the virus with the host cell.After the binding of S1 subunit to ACE2 through receptor binding domain(RBD),S2 subunit fusion peptide(FP)is exposed and inserts into the cell membrane,and HR2 binds to HR1 to form a 6-helical bundle structure,pulling the cell membrane at the N end of HR1 and the viral membrane at the C end of HR2 together and resulting in membrane fusion.The S2 subunit of different coronaviruses are more conserved than the S1 subunit,and studies on S2 subunit can provide usefully clues for the prevention,diagnosis,and treatment of coronavirus caused diseases.Obtaining purified S2 subunit with its native antigenicity is important for the structural and functional research on S2 subunits,and the purified protein can also be used in the preparation and screening of monoclonal antibodies,laying the foundation for the design of epitope vaccines targeting S2 subunits.In this study,baculovirus expression system was used to express,secrete,and purify the S2 subunit from different coronaviruses.The main research contents and results are as follows:(1)Eight SARS-Co V-2 associated coronaviruses(Ra TG13、WIV1、GX-P5L、GD18、Civet SZ3、Civet 007、SARS-Co V、SARS-Co V-2)were selected.Bioinformatics analysis of the S proteins were performed to identify the major antigenic region in S2 subunit.The gene fragments encoding these S2 subunits were cloned into expression vectors containing different signal peptide sequences and different tags for protein detection and purification.(2)The expression and secretion of four coronavirus S2 proteins using a pQB3 vector with GP64 sp and His Tag were examined,which detected the expression and secretion of the four S2 proteins.Compared to Sf9 cells,S2 proteins were expressed and secreted in higher levels in High Five cells.However,purification of a S2 protein with His Tag using Ni-Agarose Resin revealed a low retaining.(3)Expression of the four S2 proteins using p Tri Ex vectors with Fib Hsp and m Fc Tag found that the expression levels of S2-Fc fusion proteins were increased.However,the S2-Fc from WIV1 strain was not secreted.A S2-Fc fusion protein was purified by using Protein A Agarose,but some cleaved forms of the fusion protein were detected in the purified protein.The protein cleavage could occur during the virus infected cell culture and the protein elution at low p H.(4)A pQB4-SH recombinant vector was constructed by inserting Strep Tag on the basis of pQB4 vector.This vector was used to express 8 coronavirus S2 proteins,and the S2 protein achieved good expression and secretion levels.The purification efficiency of the S2 protein with Strep Tag was significantly improved by using the Strep-Tactin XT 4Flow purification system.(5)In the study of S2 protein from bat-derived coronavirus WIV 1 strain,a mutant containing the changes of Arg at position 297 to Gln and Val at position 410 to Ile were obtained.The protein expression results revealed that the two amino acid mutations did not affect the protein expression,but significantly improved the recovery efficiency of purified protein.Structural prediction analysis showed that the two mutations resulted in the exposure of the N-terminal Strep Tag of the recombinant protein,which allowed better binding of the protein to the affinity column.(6)By BCA protein quantification,the result showed that S2 proteins from Ra TG13 and WIV1 strains had the highest yield of purified protein which were close to 4 mg/L;The S2 protein from Civet 007 had the lowest yield,but it also exceeded 2mg/L.(7)Antigenicity of the purified S2 proteins were tested using clinical human sera.Most of the sera taken before and after SARS-Co V-2 vaccine booster shot gave positive results with the 8 coronavirus S2 antigens obtained in this study,and stronger reactivities were detected with the post booster sera.The results indicate that the coronavirus S2 proteins obtained in this study has good antigenicity,and the S2 proteins from the 8 coronaviruses share some common antigenic epitopes,which have cross reactivity with the anti-serum against SARSCo V-2.In summary,an expression and purification system for the abundant production of secretory coronavirus S2 proteins has been established in this study,which lays the foundation for further researches on the structure and function of coronavirus S2 protein,screening monoclonal antibodies and designing epitope vaccines targeting S2 subunits.