| So far,the best way to prevent and control the spread of infectious diseases is still to vaccinate.Traditional live attenuated vaccines can induce a long-lasting immune response,but may cause unexpected side effects.Although subunit vaccines have high safety,they often fail to induce effective cellular immune effect.Therefore,in the development of vaccines,immune stimulators are needed to improve the immune efficacy of vaccines.Nanotechnology-based antigen display has been developed as a vaccine strategy,and antigen display particles have been shown to be effective in enhancing the immunogenicity of antigens.However,the exact method of antigen display,especially the simultaneous display of multiple antigens on the same particle,is still a problem that scientists need to solve in recent years.Affinity peptides have a broad application prospect due to its advantages of low molecular weight,high affinity,no immunogenicity,easy synthesis,good surface density,low production cost and so on.Synthetic peptides as specific biometric ligands have been studied deeply in recent years,and have been widely used in medical treatment,targeted drug delivery and protein purification.The development of molecular docking technology has provided a strong support for the design of affinity peptides with new activities.The high affinity and targeting of peptides also provide new possibilities for peptide-mediated antigen display.The purpose of this study was to investigate the application potential of affinity peptides,which were screened by virtual screening,in the establishment of antigen-display nanoparticles.The SYBYL-X2.1.1 software was used to implement molecular docking,and through experimental verification,the affinity peptides targeting Classical Swine Fever Virus E2 protein or Porcine Circovirus 2 Capsid protein were designed,screened and identified respectively.Using polylactic acid-glycolic acid polymer(PLGA)and Gram-positive enhancer matrix(GEM)nanoparticles as particle carriers,the antigen-display nanoparticles system was established through the high affinity between the peptides and the target proteins,and the immunological activities of the prepared antigen-display nanoparticles were identified.The specific contents are as follows:1.Design of affinity peptides targeting CSFV E2 and PCV2 CapThe protein crystal structures of PCV2 Cap from Protein Data Bank(PDB),and the crystal structure of CSFV E2,which modeled from the homologous crystal structure of E2 protein of bovine viral diarrhea virus by SWISS-MODEL,were analyzed,and appropriate docking active pockets were selected according to needs.By using SYBYL-X2.1.1 software,the linear peptide library with the length of 2-9amino acids was established in the mode of one-by-one extension.Then a Surflex-Dock program was used to implement molecular docking between the active pocket on the protein crystal structure and the peptide library.The docking results were evaluated by the consistency scoring function CScore,and the comprehensive score was displayed as a Total?Score value.And a create/MOLCAD module was used to generate different molecular solvent surfaces of peptides and proteins,and the interactions between peptides and proteins were analyzed.Finally,48 peptide sequences targeting CSFV E2 and 55 peptide sequences targeting PCV2 Cap were selected for synthesis and subsequent experimental verification.2.Screening of the best peptide targeting CSFV E2 and PCV2 CapEnzyme-linked immunosorbent assay(ELISA)and surface plasmon resonance assay(SPR)were used to identify the affinity and specificity of synthetic peptides to proteins.5 dominant peptides with good performance targeting CSFV E2 protein were screened out,named E2-PL15,E2-PL16,E2-PL17,E2-PL22 and E2-PL23,and their K_Dvalues were 141 n M,6.74?M,705 n M,3.38?M and 90.1 n M.4 dominant peptides with good performance targeting PCV2 Cap protein were screened out,named Cap-PL4,Cap-PL5,Cap-PL8 and Cap-PL16,and their K_Dvalues were 412.9n M,105.3 n M,790.2 n M and 650.8 n M.These results showed that all the peptides had affinity interactions with proteins.Then the peptides were conjugated to the NHS agarose magnetic beads,to verify the binding ability of different peptides to the target proteins.Based on the results,the best affinity peptides h Ig G-PL9,E2-PL23 and Cap-PL5 were screened out for the subsequent experimentals.3.The optimal conditions for peptides binding proteinsPeptides and proteins interacted through non-covalent bonds,and the changes of solution environment will lead to the enhancement or decrease of the affinity between peptides and proteins,so it was necessary to screen the optimal conditions for peptide binding proteins.The optimal conditions of E2-PL23 and Cap-PL5 binding to their target proteins were determined by changing the p H concentration and salt ion concentration of the PBS solution.The results showed that CSFV E2 bound best in a PBS solution with p H 6.0-10.0 and Na Cl concentration 0.1-0.2 M,and Cap-PL5 and PCV2 Cap bound best in a PBS solution with p H 8.0-9.0 and Na Cl concentration0.2-0.3 M.4.The establishment of system of the antigen-display nanoparticlesAs specific biometric recognition ligands,affinity peptides can perform many biological functions by binding to target proteins.Based on the specificity of affinity peptide binding proteins,this study established a system of antigen-display nanoparticles,which could display protein antigens accurately.First,the exact locations where the E2-PL23 bound to the E2 and the Cap-PL5bound to Cap were determined.It was very important for accurate dispay of the antigens.The key sites of peptide-binding protein determined by computer simulation were mutated,and the affinity between peptides and the mutant proteins were identified.The key sites of the binding of E2-PL23 to CSFV E2 were Asn-997 and Tyr-999,and the key sites of the binding of Cap-PL5 to PCV2 Cap were Arg-181 and Gln-183.Then the peptides were conjugated with PLGA and GEM by EDC/NHS method,and after incubated with the proteins,antigen-display nanoparticles were constructed.Characterization of these nanoparticles showed that 4 single-antigen display nanoparticles PLGA-E2,PLGA-Cap,GEM-E2,GEM-Cap and 3multi-antigen display nanoparticles PLGA-E2+Cap and GEM-E2+Cap were successfully constructed.Subsequently,the effects of antigen-display nanoparticles to the toxicity,uptake and cytokine expression of APCs were detected.The results showed that both PLGA groups,including PLGA,PLGA-E2,PLGA-Cap and PLGA-E2+Cap,and GEM groups,including GEM,GEM-E2,GEM-Cap and GEM-E2+Cap,have no toxicity to APCs,and the GEM groups could promote the growth of RAW264.7 with high concentrations.And both PLGA groups and GEM groups could promote the phagocytosis of RAW264.7 and could promote the inflammatory action of APCs without causing inflammatory damage,and promote the differentiation and maturation of dendritic cells.Finally,mice were immunized with antigen-display nanoparticles containing the same amount of antigen proteins,and the antibody levels and neutralizing antibody levels in the serum of the immunized mice were measured.The results revealed that both antibody levels and neutralizing antibody levels stimulated by antigen-display nanoparticles were higher than that induced by the protein groups extremely.So,these antigen-display nanoparticles had excellent immunological activities.To sum up,the method of screening affinity peptides with novel activities based on molecular docking technique was very reliable.The affinity peptides,screened out by molecular docking,were with high affinity and specificity,and they had achieved good results in the applications of Ig G enrichment from complex environment and assembly of antigen-display nanoparticles.It provided a new idea for protein purification and subunit particle vaccine development based on affinity peptides.In addition,our peptide-mediated antigen display nanoparticles also enable multiple antigens to be displayed on the same nanoparticle,providing support for the development of multi-conjugate vaccines. |
PDF Full Text Request