| Objective:The annual epidemiology of seasonal influenza and the potential threat of pandemic flu was a great challenge for human health and social stability.Influenza virus possesses the characteristics of high mutation rate and gene reassortment,which limits the development of broad-spectrum influenza vaccines and the development of antiviral dr?gs.The main antigens contained in the currently used influenza vaccines(split,inactivated)was the HA and NA viral protein.Because of antigen shifts and conversions,it's necessary for us to prepare annual seasonal influenza vaccine according to WHO-predicted strains.The delay of this strain screening and preparation process can not meet the rapid response and broad spectrum to prevention and control required by the health authorities of different countries.Therefore,the screening and development of subunit vaccines with influenza virus-conservative antigens,and the technology platform for antigen presentation via structural biology and nanotechnology have become important research regions for influenza vaccines.In this field,the extracell?lar portion of the viral matrix protein M2 has good conservation and good antigenicity in the influenza A virus,which has become one of the candidate antigens for the general influenza vaccine.At the same time,Helicobacter pylori ferritin(Ferritin)can self-fold into octahedral spherical nanoparticles composed of 24 subunits,which has good antigenicity and immunogenicity after fusion expression of specific pathogen gene fragments.This project uses the extracell?lar portion of the influenza virus transmembrane protein M2 for this subject.Fusion expression under the guidance of ferritin protein,and self-assembly of nanoparticles,provides the new orientation for experimental vaccine researches.Methods:The pcDNATM3.1-M2e-ferriti,SNAP-M2e-ferritin,pcDNATM3.1-6His-M2e-ferritin eukaryotic expression vector was constructed,and the 293T cells were tansfected and c?ltured for 48 hours.The expression and purification of fusion proteins were detected using denaturing or non-denaturing SDS-PAGE and immunoblot techniques.The intracellp.lar and purified morphology of the expressed protein and its self-assembled particles were observed by excitation confocal microscopy and transmission electron microscopy.The antigenicity and immunogenicity of the nanoparticles were evaluated using Balb/c mice aged 4-6 weeks.10?g/immunization and 2(interval 14 days)immunization procedures were performed for subcutaneous injection of M2e particle antigen,and after complete immunization,2000 HI titer of H1N1 viral flu was inoc?lated intranasally and challenged.The body weight changes of the mice after challenge,the changes of lymphocytes in the spleen,and the proliferation of antigen-specific T/B cells were analyzed.Res?lts:Thro?gh denaturing and non-denaturing Western blot techniques,immunofluorescence,electron microscopy,etc.,it was determined that the ferritin-based influenza A virus M2e antigen nanoparticles were successf?lly constructed.It was found that nanoparticles with uniform size and structure were formed in the cells.Neutralization assays and inhibition of hemagglutination of the serum of the mice immunized with the protein confirmed the immunogenicity and protective properties of the constructed nanoparticles.Conclusion:The constructed nanoparticles of the influenza virus M2e antigen based on ferritin can stim?late mice to produce highly efficient neutralizing antibodies with good antigenicity and immunogenicity.In summary,our research provide a good theoretical and technical foundation for building an autonomously designed structural target and establishing an efficient,simple,rapid,and broad-spectrum influenza subunit vaccine production platform. |
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