| Bacterial diarrhea caused by infection with pathogenic microorganisms is a common health problem in many developing countries.In particular,diarrhea in infants and young children under the age of 5 and traveller's diarrhea are currently not available.Clinical studies have shown that a single consensus vaccine cannot cope with the diversity and polymorphism of pathogens,leading to an inability to cause overall protection against bacterial diarrhea.Therefore,our main research content is to develop new safe and effective combined diarrhea vaccine preparations.The nucleic acid vaccine as a combined vaccine carrier has the advantages of facilitating multi-antigen combined immunization,simple and rapid preparation process,and simultaneously inducing humoral immunity and cellular immunity,but has disadvantages such as poor immune response in large animals and humans.Therefore,the purpose of this study was to develop a multivalent DNA vaccine for both ETEC and Shigella pathogens.The research content mainly involved the design of multivalent DNA vaccines and the selection of highly efficient expression vectors.Therefore,the following work was carried out in this study.And achieved corresponding results:Firstly,the main pathogenic factors of ETEC pathogens LTB,CfaE,CssA,S.Sonnei pathogenic bacteria IPaB,using bioinformatics software to screen B and T cell epitopes,select 16 The dominant epitopes include linear B epitopes(4),nonlinear B epitopes(4),HTL epitopes(4),and CTL epitopes(4).Rational design of MEG and MB multi-epitope sequences,through 3D Structural simulation and antigenic prediction resulted in a multi-epitope sequence with an antigenic index of up to 0.94.Next,a replicon vector pSFV capable of efficiently expressing a foreign protein was selected,and recombinant plasmids pSFV-MEG and pSFV-MB were constructed as DNA candidate vaccines.In vitro cells are subjected to preliminary detection of immunofluorescence and apoptosis ability.The results showed that MEG and MB antigenic proteins were expressed in eukaryotic cells.Finally,DNA vaccine preparations were prepared using liposome-polymer hybrid nanoparticle delivery systems(PLNPs)and immunological effects were evaluated in BALB/C mice.The results of the experiment showed that the candidate vaccine pSFV-MEG/PLNPs,after immunizing the mice three times at a dose of 100 ?g/time,induced significant IgG antibody levels in the serum of mice compared with PBS and blank PLNPs negative control group(P<0.01),and the vaccine vector pSFV induced a higher serum antibody level than the common vaccine vector,with significant difference(P<0.05);the proportion of mouse spleen T lymphocyte CD4+T cells increased,CD8+T cells The proportion decreased and the ratio of CD4+T/CD8+T increased,indicating that the candidate vaccine pSFV-MEG/PLNPs can stimulate the cellular immune system of the body.Therefore,pSFV-MEG/PLNPs have been shown to be candidates for vaccine against epidemic diarrhea.In summary,this study used bioinformatics tools to screen B and T cell epitopes,rationally designed to construct a replicon DNA vaccine expression vector(pSFV-MEG),and applied a liposome-polymer hybrid nanoparticle delivery system.The corresponding DNA vaccine preparations were prepared and tested by antibody level and cellular immunity level.The results showed that the pSFV-MEG/PLNPs vaccine can induce strong humoral immunity and cellular immunity,so it can be used as a potential candidate vaccine to solve bacterial diarrhea.Provide new design ideas and research foundations. |
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