Screening And Identification Of Monoclonal Antibodies And Antiviral Affinity Peptides Against Goose Astrovirus Spike Protein

As a new pathogen,Goose astrovirus(GAst V)can cause systemic visceral gout in goslings,showing high infection rate and high mortality.Since the outbreak in 2016,it has brought serious losses to China’s goose industry.Spike protein is located on the surface of virus particles.As an important antigen protein,Spike protein can induce host immune response and mediate the binding of virus particles to cell surface receptors.Therefore,spike protein is an important target for diagnostic reagent development and drug screening.1.Expression and purification of GAst V Spike proteinFirstly,the constructed p ET-28a-Spike plasmid was transformed into E.coli BL21 competent cells.The positive strains with correct sequence were obtained by PCR and sequencing.After optimizing the prokaryotic expression conditions,IPTG with a final concentration of 1 m M was added at 16 ℃ for overnight induction.SDS-PAGE showed that soluble Spike protein was successfully expressed.Next,Spike protein was purified by nickel ion affinity chromatography.Identified by SDS-PAGE and Western-blot,Spike protein with high purity(95%)and high concentration(1.5 mg / m L)was obtained.2.Preparation of monoclonal antibody against GAst V Spike proteinBALB / c mice were immunized with Spike protein,cell fusion and subcloning.Three positive monoclonal cells were screened by ELISA and IPMA,which were 1A5,2B4 and12E6 respectively.Western-blot and IFA showed that the three monoclonal antibodies reacted well with Spike protein,and 2B4 and 12E6 reacted with GAst V.The results of subtype identification showed that monoclonal antibodies 12E6 and 2B4 were Ig G2 b subclass and monoclonal antibody 1A5 was Ig G2 a subclass;All light chains are κ Chains.The antibody titer was above 1: 51 200.25 overlapping peptides of 15 amino acids(including the overlapping amount of 7 amino acids)were synthesized according to the Spike sequence of GAst V XX strain.The results of ELISA and Dot-blot showed that the epitope recognized by Mc Ab 1A5 was(EP16)ELRNRLNIADGDYVI,and the epitope recognized by Mc Ab 2B4 was(EP21)AGDSNPGETFQNFKM polypeptide,all of which were linear B-cell epitopes.3.Virtual screening of GAst V Spike protein affinity peptidesTaking the structure of TAst V-2 Spike protein as the template,the homologous modeling was carried out by SWISS-MODEL,and the three-dimensional structure of GAst V Spike protein was obtained.Based on the computer virtual screening technology of molecular docking technology,SYBYL software is used to select the C-terminal of Spike protein as the active pocket to establish the polypeptide library.Then use Surflex-Dock/SYBYL to dock the polypeptide ligand in the library with the selected pocket.Finally,the docking results were comprehensively evaluated by the consistency score function(Cscore),and a total of 30 peptides specifically recognizing GAst V Spike protein were selected,which laid the foundation for the follow-up antiviral research.4.Screening and identification of antiviral affinity peptide of GAst VThree peptides that inhibit the proliferation of GAst V were preliminarily screened by IPMA.Combined with the cytotoxicity results of the peptides,the polypeptide AP21 was finally screened.Affinity identification showed that polypeptide AP21 could specifically bind to spike protein.The results of Western-blot,IFA and TCID50 showed that the affinity peptide AP21 had significant antiviral activity against GAst V,which laid a foundation for the development of GAst V antiviral drugs.The results of antiviral target analysis showed that the target of AP21 was located on the side of Spike protein structure and had a specific spatial conformation.