Rational Design,identification And Application Of Affinity Peptide Ligands Of Porcine Circovirus Type 2 Cap Protein

Porcine circovirus type 2(PCV2)is the main pathogen of porcine circovirus-related diseases(PCVAD),which has brought huge economic losses to the pig industry all over the world.Vaccination is the main means to prevent and control PCVAD.At present,PCV2 vaccines mainly include inactivated vaccines and subunit vaccines.PCV2 Cap,as the main effective component of these two types of vaccines,can be self-assembled into virus-like particles(VLP),to induce neutralizing antibodies with good antigenicity.The inactivated vaccine is produced by PK15 cells,but the virus titer is low the production cost is high.The inactivated vaccine is produced by PK15 cells,but the cost is high,the virus titer and antigen content are low.In the practical production process,the content and purity of the antigen determines the quality of the vaccine.Subunit vaccine is expressed and prepared by E.coli or baculovirus-insect cell system,which has attracted more and more attention because of its high safety.The main problem of PCV2 subunit vaccine is that antigen protein purification process is complex and at the primary purification level.In order to solve this problem,there is an urgent need for a fast and efficient purification method.This study carried out a series of experimental studies on PCV2 Cap protein affinity peptide ligands,which are divided into the following four parts:1.Rational design of affinity peptide ligand for PCV 2 Cap proteinHigh-resolution structures are possible for computer-aided design based on three-dimensional models of target proteins.In this study,a molecular docking virtual screening technology was used to construct a virtual peptide library with a length of 2-9amino acid residues.The peptide in the library were molecularly docked with specific sites on the PCV2 Cap protein,and the interaction force of the complex was analyzed by the SYBYL/MOLCAD module;Then,the molecular simulation results were comprehensively evaluated and analyzed using the Cscore value of the consistency score function.Finally,67 peptide sequences with specific recognition of PCV2 Cap were designed in order to provide help for the purification of PCV2 Cap protein and the design of new vaccine.2.The screening and identification of PCV2 Cap protein affinity peptide ligandsIn this study,soluble recombinant PCV2 Cap protein was successfully expressed in E.coli,and purified by Ni magnetic beads to obtain PCV2 Cap protein with high purity and biological activity,which provided a material basis for the screening and identification of affinity peptide ligands.Qualitative screening and rescreening by ELISA identified 13 peptides(named L1-L13)that reacted well with PCV2 Cap protein from 67peptide sequences,and their affinity and specificity were further characterized.The results showed that 5 candidate peptides:L4,L7,L9,L11,L13 had high affinity and specificity for the PCV2 Cap protein.Finally,LSPR quantitative detection of the K_Dvalues of affinity constants of 5 candidate peptides.The results showed that the K_Dvalues of the L4,L7,L9,L11,and L13 peptide sequences were 1.23?M,1.03?M,6.71?M,103 n M,and 9.97?M,respectively,indicating that the five candidate peptides have different degrees of affinity binding to the PCV2 Cap protein.The study showed that the method based on the combination of molecular docking virtual screening technology and in vitro experimental verification can improve the accuracy of screening peptide sequences and provide high-affinity peptide ligands for the subsequent preparation of affinity adsorbents.3.Preparation of affinity peptide ligand adsorbent and optimization of purification conditionsIn order to solved the problem of PCV2 Cap purification in parctical production,this chapter will carry out preliminary identification of PCV2 Cap protein purification from the 5 candidate peptides selected in the previous chapter through in vitro experiments.The better performance L11-DYWWQSWE was used as the best peptide ligand to couple with NHS agarose magnetic beads(Na MB)to prepare Na MB-L11affinity adsorbent.In the process of purification,the effects of liquid conditions(p H and salt concentration)and solid phase properties(ligand density)of affinity adsorbents on the purification of PCV2 Cap protein were studied,and also optimized the elution conditions.The experimental showed that the best purification conditions of Na MB-L11adsorbents were p H 9.0,Na Cl 250 mmol/L,ligand density 1.25 mmol/L.The purity of PCV2 Cap protein obtained in one-step purification was 98%and recovery rate was 90%.4.The construction of purified self-assembled PLGA-PCV2 Cap nanoparticleNew nanoparticle vaccines have made great progress in recent years.Among them,the study of the polymer material PLGA as a vaccine delivery vehicle has attracted the most attention.In this experiment,the affinity peptide L11 was covalently coupled to the carboxyl-PLGA(PLGA-L11).Then,the recombinant PCV2 Cap protein supernatant was added to its incubation and binding.Through simple repeated centrifugation,it can be purified and enriched to self-assembled PLGA-PCV2 Cap nanoparticles.The results of in vitro studies showed that PLGA is non-toxic to DC2.4 cells and RAW264.7 cell at the concentration of 200?g/m L and 100?g/m L,and can effectively promote the uptake of PCV2 Cap protein by APCs.In mouse experiments,compared with PCV2 Cap in solution state,PLGA-PCV2 Cap can effectively stimulate the body to produce higher antibody levels,and the titer of neutralizing antibody is also significantly increased.This study proposed a new vaccine construction strategy based on affinity peptides.The PLGA nanoparticles can be used to display the purified and enriched PCV2 Cap antigen array at high density,which can effectively improve the antibody level of the body.