The Rational Design And Application Of Affinity Peptide Of Targeting Classical Swine Fever Virus E2 Protein

Classical swine fever(CSF)is characterized as an infectious disease with high pathogenicity and mortality rates which caused by Classical swine fever virus(CSFV).CSF is epidemic with regional distribution in China.Due to unscientific immunization,uneven quality of the vaccine and irregular feeding conditions,it is still a pathogenic threats to the livestock farm in our country.E0,E1 and E2 are the three glycoprotein of CSFV.E2 represents the main target for neutralizing antibodies and the receptor-binding protein of CSFV,which is able to inhibition infection.So,the further study of E2 is of great significance to explore the infection mechanism of CSFV and new vaccine design.With the rapid development of computational molecular simulation technology,especially the wide application of molecular docking method,it provides efficient approach for the peptide screening and makes the research of protein-ligands interaction more thorough.In terms of peptide screening,it is convenient,quick,low-intensity,high-efficient and time-saving.In this study,based on the homology models,we utilized molecular docking technique to design novel peptide motifs that bind to CSFV E2 protein.The affinity of binding E2,inhibitory activity in CSFV infection and the efficiency as affinity purification was identified,provided scientific guidance and new ideas for the research on antiviral infection and CSFV vaccine development.1 The rational design of affinity peptide of CSFV E2With the analysis of more protein crystal structure,computer aided peptide design technology represents the rapid development.Based on the molecular simulation docking and virtual screening technology,a series of random peptide combinations with different lengths were set up.Then,the docking simulation were performed on specific space between the peptides and E2,and the interaction force of the complex were analyzed by MOLCAD module.Last,a series of peptides targeting CSFV E2 were design in the base of evaluation and analysis of scoring function.It may be helpful for the research on antiviral infection and CSFV vaccine development.2 The screening and identification of affinity peptide of CSFV E2In this work,firstly,computational-derived were synthesized,and the affinities and specificities binding with E2 were identified and verified by ELISA and SPR test.The results showed that eighteen peptides appeared to bind with different affinities to E2,and P86,P88,P708,P704,P92 and P90 had the higher KD value with E2.Their KD values were 1.49nM,3.88 nM,4.72 nM,4.84 nM,49.4 nM and 50.8 nM,respectively.These indicates that it is feasible that computational designing and screening the high affinity peptides via docking molecular,may be provides reliable data and materials for the later experiments.Secondly,in order to investigate the importance of local method and global method on protein-ligand docking,the relevance of the KD values and the docking score values from GalaxyPepDock and FlexX/SYBYL programs,were analyzed and evaluated.The results showed that a moderate correlation coefficient was represented between KD values and CScores from FlexX/SYBYL.The Kendall and Spearman coefficient were-0.637 and-0.767,respectively.These demonstrate that fully considering the flexibility of is better than searching for more potential binding sites on the target protein surface while performing peptide-protein molecular docking.These data provide reasonable evidence for the molecular design of peptides and guidance for the functional assignment of protein.3 Antiviral activity of affinity peptide targeting CSFV E2 proteinBlocking the interaction of virus and host cell is considered one of the most effective ways to inhibit viral infection.Based on six high affinity peptides targeting CSFV E2 identified in the former chapter,the activity to inhibit CSFV infection was further assessed.CSFV was incubated with peptide and then infected PK-15cells.The results showed that six peptides could inhibit CSFV infection on PK-15 cells,one of which P88,P86 and P708 had the highest inhibition,up to 80%.PK-15cells were blocked with peptide and incubated with CSFV.The results showed that six peptides could not inhibit CSFV infection.These indicates the affinity peptides of targeting E2 can't inhibit CSFV infection by blocking the receptor,and further confirms that the antivirus target of affinity peptide is on the surface of the virus instead of the cell receptor.These results provided a new thread of antiviral therapy and drug design for CSFV.4 The application of affinity peptide of targeting E2 in the preliminary study of new CSFV vaccineDue to the importance of E2 as a target for neutralizing antibodies,it is still the focus of new submit CSFV vaccine development.In this study,on the base of the designed peptide by molecular docking and virtual screening in the former chapter,further tests were investigated.Then,the peptide was immobilized on the magnetic and successfully purified E2 from crude rice protein.Finally,the rapid method purification of rice E2 protein was established.Mouse immune antibody evaluation showed that 14 days after the booster,CSFV-specific antibodies were detected in the mice immunized with 10?g and 25?g of purified E2.With the increase of time,the antibody blocking rate could reach 86.18%and 90.68%in the 42 days after inoculation,respectively.The generation of the neutralizing antibodies by purified E2 stimulating were much better than controls.These indicates that the peptide of targeting E2 as a tool for purification is efficient and reliable.This novel method of design peptide may provide broader platform for protein-ligand screening,and powerful tools and new thought for CSFV vaccine design.