A Preliminary Study On The Production And Function Of SARS-COV-2 Nanosubunit Vaccine

The severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）caused coronavirus disease 2019（COVID-19）pandemic and serious global public health risk.RBD has multiple antigenic epitopes and is a key target for vaccine research.It is important to master the mutation of prevalent strains and develop safe and effective vaccines for the prevention and control of COVID-19.Subunit vaccines have the advantages of high safety and easy production and preparation.In this study,RBD and RBD-Ferritin nanosubunit vaccines against SARS-CoV-2 were prepared by bioinformatics analysis,prokaryotic expression,affinity chromatography,and serological assay,and their immunogenicity was initially investigated.The specific research contents are as follows:1.Sequence characterization of novel coronavirus RBD gene and construction of prokaryotic expression vector:amino acid multiple sequence comparison between the original SARS-CoV-2 strain and the RBD of Delta,Alpha,Beta,Zeta,Lota,Epsilon,Kappa,Mu,Gamma,and Eta mutant strains,and the homology was 98.65-99.55%,and 92.82-93.27%with the three Omicron mutant strains.The physical and chemical properties of RBD and RBD-Ferritin fusion proteins showed that they were highly hydrophilic under conventional conditions,and their secondary structures were mainlyα-helical and irregularly convoluted.The nucleotide sequence of RBD was optimized and cloned into p ET-28a vector according to the codon usage preference of E.coli,and then the p ET-28a-RBD-Ferritin expression vector was successfully constructed by PCR amplification of Ferritin and enzymatic ligation experiments.2.Expression,purification and replication of RBD and RBD-Ferritin fusion proteins:RBD and RBD-Ferritin fusion proteins were obtained by IPTG-induced expression in E.coli BL21,and their expression forms were analyzed using SDS-PAGE electrophoresis.The results showed that bands of RBD and RBD-Ferritin fusion proteins were detected in inclusion bodies and total proteome with the expected size,and Western-blot identification showed specific protein bands at 25 k Da and 41 k Da,indicating that both proteins were expressed in bacteria mainly as inclusion bodies.A large amount of high-purity target protein was obtained by Ni²⁺affinity chromatography purification,and the protein was gradient-replicated using a desalting column,and the SDS-PAGE electrophoresis results showed that active protein was obtained.The nanoparticle size results showed that the recombinant protein particles were uniform in size,and the protein stability results showed that the protein was stored at 25℃for 7 days without degradation.3.Immunogenicity study of RBD-Ferritin nanosubunit vaccine:Thirty-six 4-week-old female BALB/c mice were selected,and the purified RBD and RBD-Ferritin fusion proteins were used as antigens.The first immunization doses were 15μg and 25μg for the single-dose group,respectively,and the adjuvant group was injected subcutaneously with aluminum hydroxide in a 1:1 ratio,and the doses were halved for three subsequent booster immunizations.At weeks 1,2,3,4 and 10,mice were subjected to orbital vein blood collection and serum isolation for specific Ig G and Ig M antibody potency testing.The specific Ig G and Ig M antibody potencies of the experimental group were significantly higher than those of the control group at week 1.The Ig G antibody potencies of the RBD-Ferritin group were significantly higher than those of the RBD group at the first 4weeks,and the Ig M antibody potencies were significantly different at weeks2 and 4.The Ig G antibody potency in the adjuvant group was significantly higher than that in the single-dose group at weeks 2 and 3,but the addition of adjuvant did not affect the Ig M antibody potency.Higher titers of Ig G and Ig M antibodies were still detected in the experimental group at week 10.In this study,we demonstrated that RBD is stable and conserved,RBD and RBD-Ferritin fusion proteins can be expressed in large amounts in E.coli,and the recombinant proteins obtained by affinity chromatography can induce the production of specific serum Ig G and Ig M antibodies in mice.Increasing the number of immunizations induced a sustained immune response,and the RBD-Ferritin fusion protein induced a significantly higher Ig G potency than the RBD protein,and the addition of aluminum hydroxide adjuvant further enhanced the immune response.This study provides a reference for the preparation of SARS-CoV-2 vaccine by applying Ferritin nano-cage structure,and lays the foundation for studying the pathogenesis and serological detection of novel coronaviruses.