| Vaccine is of great significance in the prevention and control of infectious diseases.Traditional inactivated vaccines and live attenuated vaccines contain unnecessary antigen loads,which may reverse toxicity or cause unnecessary immune responses.For safety reasons,people began to use the necessary components of pathogens such as peptides,proteins,membranes,polysaccharides,toxins,etc.to prepare subunit vaccines.Compared with traditional vaccines,subunit vaccines have a single component and high safety,but their immunogenicity is often low,and they cannot produce a high level of immune response when used alone.Participation fails to form immune memory.With the development of nanotechnology,nano-vaccine prepared by loading antigen with nano-delivery carrier can enhance the immunogenicity of antigen,stimulate high-level humoral and cellular immune responses,and generate immune memory.Based on different materials,the carriers used in nano-vaccine mainly include lipid nanoparticles,protein nanoparticles,polymer nanoparticles,inorganic nanoparticles and so on.Among them,protein nanoparticles have great application potential due to their good biocompatibility and safety.In the protein nanovaccine delivery system,the antigen and the delivery carrier are often coupled by fusion expression,but this method may cause structural interference between the delivery carrier and the antigen,which affects the stability of the delivery system and the final immune effect.In addition,the fusion preparation of carrier and antigen is not conducive to the establishment of a universal vaccine chassis system.In our study,We used biotin and monovalent streptavidin(m SA)as binding tags,exploiting the high affinity between the two to couple the antigen to the delivery vehicle.An orthogonal,modular,and highly versatile self-assembled protein nanoparticle chassis was established.Its stability was checked and the binding of the carrier to the antigen was assessed.The specific research contents are as follows:Our previous studies have demonstrated that CTBTri,which can be self-assembled into nanoparticles(NP),can be loaded with various antigens through fusion expression or glycosylation system,and stimulates strong stimulation by promoting lymph node drainage,antigen presentation,T cell proliferation and differentiation,and B cell maturation.humoral and cellular immune responses.Further,we hope that nanoparticles can be used independently as a chassis to realize the modular design of delivery system.However,we found that the yield of CTBTri alone was extremely low as a chassis,and considering the high hydrophilicity of polysaccharide glycan chains,we hoped to improve its solubility and yield by glycan modification of NP.In order to avoid the polysaccharide chain causing polysaccharide-specific immune response,we used theλ-RED homologous recombination system to construct a rfc and waa I double mutant S.flexneri 2a 301 strain.We first knocked out the O-antigen polymerase gene rfc to prevent the repeat unit(RU)from polymerizing into OPS,so that only one RU remained on the lipid A core,and then knocked out the O-antigen polymerase gene waa I,and further The transfer of RU to the lipid A core was prevented,and the RU was freed in the periplasm,and the strain was named 301DRW.Finally,the existing plasmid p ET28a-Pgl L-CTBTri4573 in the laboratory was transferred into 301DRW,in which Pgl L is an O-glycosyltransferase,which can transfer pathogenic polysaccharides to the glycosylation site serine,and 4573 is the natural substrate of O-glycosylation Pil E.The29 amino acid sequences before and after serine 63 were induced and expressed to obtain NPs modified with short sugar chains(NP-RU),and the RU was identified by LC/MC-MC.At the same time,it was found that the expression level was significantly higher than that of NP.Purified by nickel column and molecular sieve,NP-RU with a purity of more than90%was obtained,and its particle size was about 20-30 nm observed by transmission electron microscope.Dynamic light scattering(DLS)results showed that it was a monodisperse particle with a hydrated particle size of about 30 nm.The DLS results were consistent with the electron microscope results,demonstrating the successful self-assembly of NP-RU.To confirm the stability of the nanoparticles,we placed the NP-RU at 37°C for 48 h,performed DLS measurement again,and found that the size was stable.The characterization results show that the obtained NP-RU particle size is consistent with the theory and can exist stably,and can be used for further experiments.The obtained NP-RU was used to subcutaneously immunize mice,and the plasma cytokine profiles at different times after immunization of mice were analyzed.It was found that both Th1 and Th2-related cytokines were increased,indicating that the immune response of cells and humors was enhanced.In addition,we measured serum titers by subcutaneously immunizing mice,and found that NP-RU did not stimulate the body to produce specific antibodies against RU,confirming that NP-RU can be used as a delivery vehicle.To achieve conjugation of the delivery vehicle to the antigen,we chemically biotinylated NP-RU.After removal of excess unbound small molecule biotin by dialysis,we characterized the biotinylated nanoparticle Bio-NP-RU.Coomassie brilliant blue staining and Western blot detection showed that Bio-NP-RU was slightly shifted upwards than the NP-RU band,which proved that Bio-NP-RU had been successfully biotinylated,and the biotinylation efficiency reached 100%.At the same time,the particle size of nanoparticles before and after biotin analysis was compared,and the results showed that Bio-NP-RU was still a monodispersed nanoparticle,suggesting that biotinylation would not affect the self-assembly of protein nanoparticles.So far,we have successfully obtained the biotin-labeled short sugar chain modified protein nano-delivery carrier chassis.We fused and expressed m SA at the N-terminus of the receptor binding domain(RBD)of the S protein of SARS-Co V-2,constructed a plasmid pc DNA3.1-m SA-RBD,and transferred it into HEK293 cells for expression and purification to obtain m SA-OPS.In addition,in order to obtain the polysaccharide antigen that can be coupled with the nano-delivery vector,we designed the plasmid p ET28a-Pgl L-m SA4573,in which m SA was fused and expressed at the N-terminus of4573.Transferred into 301DWP(deletion of O-antigen ligase gene waa I and virulence plasmid),induced expression and purification to obtain Shigella 301 polysaccharide antigen m SA-OPS.To verify the binding ability of Bio-NP-RU to m SA-antigen,the affinity(K_d)between Bio-NP-RU and m SA-RBD was 2.01 n M detected by surface plasmon resonance(SPR).At the same time,we also used ELISA,Western blot and native electrophoresis to prove that Bio-NP-RU can bind to m SA-RBD with high affinity.High affinity binding between Bio-NP-RU and m SA-OPS was also demonstrated by the same method.These results demonstrate that our biotinylated nanoparticle chassis is capable of loading m SA-antigens with high affinity.In this study,an orthogonal,modular,and highly versatile self-assembled protein nanoparticle chassis was obtained by engineering Shigella to modify short sugar chains by O-glycosylation.By modifying biotin on the surface of nanoparticles,it can be loaded with antigens fused to the m SA tag.The vector and antigen in this system are separately purified and then assembled,which is not limited by the expression system and has a wide range of applications.The use of O glycosylation to produce m SA-containing polysaccharide antigens means that preventive nanopolysaccharide conjugate vaccines for different bacteria can be prepared.In addition,a universal chassis can be prepared in advance and assembled with various pathogenic bacteria antigens according to requirements,shortening the response time to diseases.It provides a new idea for the modular preparation of nano-vaccine.In the future,we will further optimize the nanoparticles and evaluate the immune protection of vaccines. |
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