Preparation And Evaluation Of Different Domain Antigen Of Sars-CoV-2 S Protein Containing Intramolecular Adjuvant

The epidemic caused by the new coronavirus（SARS-Co V-2）has swept the world since the end of 2019.As the virus continues to mutate,the epidemic situation at home and abroad is severe,vaccine plays a leading role in the prevention of virus infection.Enhancing the effectiveness and safety of COVID-19 vaccine is the key to further improve the protection rate of vaccine.As one of the main routes of vaccine development,recombinant subunit vaccine has clear antigen structure and amino acid sequence,and can be modified by genetic engineering.However,the antigen molecular weight of subunit vaccine is small,and the immune effect is weak.Improving its immunogenicity is the main research direction of subunit vaccine.SARS-Co V-2 S protein contains major neutralizing epitopes and is the preferred target protein for new crown subunit vaccine development.It has been reported that tetanus toxin peptide（TT peptide）is a universal T cell p2 epitope(TT_830-843),which is often used as an intramolecular adjuvant and connects with the target protein in an appropriate way,which can significantly improve its immune effect.In this study,intramolecular adjuvant TT_830-843 and SARS-Co V-2 S protein were combined in series with different domains,and the fusion protein containing intramolecular adjuvant was prepared by prokaryotic expression system.The immune response（humoral and cellular immune response）of the fusion protein containing intramolecular adjuvant was evaluated by experimental animals to explore the effect of intramolecular adjuvant TT_830-843 on antigenic immunity.In this study,the S protein sequence of SARS-Co V-2 was analyzed and three regions:NTD,RBD and S2 subunit were truncated,and the intramolecular adjuvant was connected with the intercepted sequence to construct a recombinant plasmid and transformed into BL21（DE3）engineering bacteria,named S-TT1,S-TT2 and S-TT.The induction temperature and inducer concentration of the three bacteria were optimized respectively.The results showed that the three bacteria were expressed in inclusion bodies.The expression level was higher when induced at 37℃for 4 h and the concentration of inducer was 0.5mmol/L.The purity of the three recombinant proteins can reach more than 90%after purification by ion exchange chromatography;the three inclusion body proteins were renatured by dialysis,and the antigenicity of the target protein was proved by Western blotting and Dot-ELISA tests;the endotoxin content of the target protein was reduced to less than 10 EU/m L by anion exchange chromatography and Triton X-114 method.The results of immune experiment showed that through the comparison of the adsorption rate and serum positive rate of aluminum adjuvant with different doses of three antigens,the optimum immune dose of the three antigens was 50μg.The specific antibody level of mice immunized with S-TT1,S-TT2and S-TT3 was significantly different from that of PBS group.Pseudovirus neutralization test showed that the neutralization titer of S-TT2 was 1:30,the neutralization activity was higher,and the dominant antigen was S-TT2.The immune effect of dominant antigen containing intramolecular adjuvant was further evaluated.The results of long-term stability and accelerated stability showed that the prepared S-TT2 immune preparation was stable at 4℃for 12 months and 37℃for 10 days.The results of ELISA antibody titer showed that the specific antibody titer of the dominant antigen S-TT2 immunized group was significantly higher than that of the S-RBD antigen immunized group,and the pseudovirus neutralization test showed that the neutralization titer of S-RBD immunized mice was 1:17,which was lower than that of the S-TT2 group.The lymphocyte proliferation test showed that the specific stimulating index of S-TT2 antigen to lymphocytes was 4.64±0.12.In the presence of intramolecular adjuvant TT peptide,the antigen promoted the secretion of IL-2,IFN-γand IL-4 cytokines,and also stimulated the production of more CD4⁺T lymphocytes in mice.To sum up,three kinds of recombinant proteins S-TT1,S-TT2 and S-TT3 with high purity and activity were obtained in this study,and the dominant antigen with high neutralizing activity was selected as S-TT2.Further study on the immune effect of S-TT2 antigen showed that intramolecular adjuvant TT_830-843 could improve the cellular and humoral immunity,which provided an idea for improving the immune effect of SARS-Co V-2 and other virus subunit vaccines.