| [Background and purpose] COVID-19 is a serious infectious disease detected and broke out in 2019.It seriously affects the economic and social development of our country and even the world.Under the epidemic,the number of infections and even deaths around the world is happening every day.China’s effective anti-epidemic policies,major investment in vaccine research and development,and free vaccination policies for all have enabled China’s epidemic to be quickly controlled.However,the epidemic has spread rapidly abroad,and many countries around the world have been affected by COVID-19,and vaccines are far from being in short supply.Even,SARS-Co V-2 variants reported around the world continue to appear,including the highly infectious Delta strain,Omicron strain and other variants that caused a second outbreak of the global epidemic.In our country,there are still some people infected with these variants.The outbreak caused by a variant of SARS-Co V-2 is showing signs of making a comeback.Therefore,in order to deal with the global vaccine inequity and the mutation of SARS-Co V-2,we designed and developed an effective nanovaccine with a simple production process and strong reassembly ability.It splices the S1 subunit of the spike protein and the envelope protein to form a fusion protein,which is coated on the PLGA nanoparticles.[Methods] The genome sequence of SAR-Co V-2 was found in NCBI gene library,and S1 sequence and E sequence were selected to splice into S1-E sequence by Linker.The designed S1-E sequence was analyzed by bioinformatics,including hydrophobicity,epitopes and tertiary structure prediction.The S1-E sequence was synthesized.The S1-E p ET-28a(+)recombinant plasmid was constructed and subjected to transformation,cloning and extraction.The extracted plasmids were verified and transferred into competent cells Rosetta(DE3).The recombinant S1-E protein was induced by IPTG and then purified.The recombinant S1-E protein was coated on PLGA nanoparticles by Double Emulsion-solvent Evaporation Technique to prepare S1-E-PLGA nanovaccine,and the characterization of nanovaccine was tested.BALB/c mice were inoculated and the immune effect of recombinant S1-E protein and S1-E-PLGA nano vaccine was evaluated.[Results] The recombinant plasmid carrying the S1-E sequence was successfully constructed and the recombinant S1-E protein was successfully expressed in the prokaryotic expression system,and the protein size was about 50 k Da.The S1-E-PLGA nanovaccine showed spherical shape under the scanning electron microscope,the loading capacity was 8.8,the encapsulation rate was 63.6%,the particle size distribution was 670±145 nm,and the Zeta potential was-22.3±6.89.After recombinant S1-E protein,recombinant S1-E protein + Freund’s adjuvant,and S1-E-PLGA nanovaccine were respectively inoculated into BALB/c mice,compared with the control group,anti-S1 and anti-E antibodies were produced.Pseudovirus neutralizing antibodies are produced.And the results show that the antibody titer produced by S1-E-PLGA is higher than that of recombinant S1-E protein+Freund’s adjuvant,and it has higher neutralizing ability.After stimulation with recombinant S1-E protein,the percentages of IL-2 and TNF-α positive T cells in each group were higher than those in the control group.Meanwhile,the mice’s mental state and weight gain were normal.[Conclusion] Recombinant S1-E protein can stimulate a good immune response in mice,and S1-E-PLGA nanovaccine can stimulate a better immune response in mice... |
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