Construction And Efficacy Of Sars-CoV-2 S Protein Multi-epitope Recombinant Protein Vaccine

SARS-CoV-2 is a worldwide pandemic and vaccines against the virus is being actively developed worldwide.The most used target antigen in the development process is the S protein.The S protein is one of the structural proteins of SARS-CoV-2,which uses the receptor binding domain(RBD)to bind to angiotensin 2.The S protein not only determines the infectivity and transmissibility of the virus,but also induces the production of high-titer specific antibodies in SARS-CoV-2 infected individuals.The process of antibody production induced by viral infection of the organism is mediated by T and B cells.The aim of this study was therefore to construct a multi-epitope recombinant protein vaccine antigen using PCR and subcloning and using animals to investigate its efficacy,based on T and B cell epitopes of the S protein screened by bioinformatic methods of prediction.The main elements include the following:Ⅰ.Prediction and screening of B-cell epitopes on SARS-CoV-2 S proteins based on bioinformaticsIn order to predict and screen B-cell epitopes on SARS-CoV-2 S protein,we first downloaded the amino acid sequence of S protein from the public website NCBI to simulate its spatial structure,analyzed the secondary structure of S protein,and initially screened B-cell epitopes based on its hydrophilicity,antigenic index and surface likelihood,and then used the tool Bepi Pred2.0 to predict the SARS-CoV-2 S protein on The region where the linear B-cell epitope is located was further analyzed and integrated to screen out the more exposed coiled peptides by comparing the two on the viewable view of the S-protein spatial structure,and the two selected peptides were B2(343-358aa)and B3(472-489aa),respectively.Ⅱ.Design,construction,expression and purification of a multi-epitope vaccine antigen E3 and E3-his for SARS-CoV-2 S proteinT-cell epitopes were screened for pre-lab results and the two T-cell epitopes selected for this study were T2(338-352aa)and T3(919-933aa).To construct the antigen E3-his,the selected T and B epitopes were linked in tandem to design and construct the antigens E3 and E3-his,the latter carrying the his-tag tag.The spatial structure was predicted by comparing B2 and B3 on E3 and S proteins(early strains).E3 possessed 9 B2 s and 4 B3 s,probing for morphologically consistent B epitopes on S proteins and arguing that the greater the number of morphologically similar ones,the more similar E3 and S proteins were.This led to the design of amino acid sequences for E3 and E3-his.To construct the genes encoding E3 and E3-his,the A and B fragments need to be constructed first.The plasmid carrying the A fragment has been successfully constructed,so the B fragment needs to be constructed for this study.The B fragment was ligated to the cloning vector by PCR to construct a cloning plasmid carrying the B fragment,and the recombinant plasmid carrying the E3 coding gene was constructed by ligating the A and B fragments using pre-designed digestion sites.The E3 and E3-his coding genes were integrated into the expression vector by subcloning technique,and the strain was cultured at 37°C in a shaker with IPTG(1m M)to induce expression.Inclusion body proteins were obtained and purified.Ⅲ.Immunization of animals with E3-his and recombinant S protein vaccines and detection of antibody levels by ELISAE3-his consists of immunogenic epitopes on S proteins.To investigate the potency of E3-his,in this study,purified E3-his and purchased recombinant S proteins were used as vaccine antigens,respectively,in combination with oil-in-water adjuvants(SA08,P111,T1,T2,T5 and T6)to form a vaccine,and antibody levels of immunized mice were measured using ELISA results.ELISA results(immune serum with E3-his antigen using E3-his packet plates)indicated that E3-his stimulated the production of specific antibodies in mice,but the results of the horseshoe crab reagent assay indicated that purified E3-his contained endotoxin,indicating that the protein has immunological activity and that the endotoxin was a natural adjuvant.immune sera)showed that sera immunized with S protein recognized E3-his and that the two had similar antigenic properties.We also investigated whether the immune cells "remember" the adjuvant and showed a delayed decline in antibody response over a week,but this needs to be explored further.In summary,E3-his is potent as a recombinant multi-epitope protein vaccine antigen and the presence of endotoxin enhances its antibody response.a specific association exists between E3-his and S proteins.sera immunized with S proteins recognize E3-his,suggesting that E3-his and S proteins have similar antigenicity and that E3-his-immunized sera have the potential to neutralize SARS-CoV-2early strains.In addition,antibody sera immunized with S protein could be used as detection antibodies to assess the quality of recombinant subunit vaccines.This study provides design ideas and a platform for the development of a multi-epitope recombinant protein vaccine against SARS-CoV-2 S protein.