Construction And Immunization Effect Study Of MSRV Vaccine Based On Phage Display Antibody Library

The rhabdovirus disease caused by Micropterus salmoides rhabdovirus（MSRV）poses a serious harm to the bass culture industry.Vaccine as an important method of protection against viral diseases can effectively prevent farmed fish from viruses.Glycoprotein ha s received a lot of attention in the course of rhabdovirus vaccine research because it contains abundant antigen epitopes,but the antigen epitopes where the MSRV glycoprotein plays a key role are still unclear.In this study,the spleen tissue of Micropterus salmoides immunized with MSRV glycoprotein was used as a source of gene information and combined with phage display technology（PDT）to construct the antibody library.High affinity antibodies were obtained by biopanning and evaluated for anti-MSRV activity.On this basis,bioinformatics techniques were used to analyze the antigen-antibody interaction sites.After identifying the key antigen epitopes,KLH-coupled peptide vaccine was constructed and evaluated for immune protection.The study results were as follows:1.Construction and biopanning of anti-MSRV phage display libraryMicropterus salmoides was immunized by intraperitoneal injection,and weekly serum antibody potency levels were analyzed by enzyme linked immunosorbent assay（ELISA）.Extraction of RNA from spleen tissue at peak serum antibody potency and reverse transcription to c DNA.The antibody heavy chain gene and light chain gene were amplified by PCR to construct an anti-MSRV phage display antibody library.The antibody library was biopanned using the solid-phase sieve elution method,followed by phage-ELISA to detect the affinity of different antibodies.The results showed that after immunization with MSRV glycoprotein（G2）,the serum antibody potency level of Micropterus salmoides increased significantly,reaching a peak at 21 d(OD₄₅₀=1.023).Agarose gel electrophoresis showed that the size of the amplified antibody heavy chain gene,light chain gene,and single chain antibody were 330 bp,330 bp,700 bp.The results of plate counting showed that the library capacity of the anti-MSRV phage display antibody library was 2.16×10⁵.After 4 rounds of biopanning,the output-input ratio increased round by round and the enrichment multiple reached 47.The results of phage-ELISA showed that 30 out of 38randomly picked monoclonal clones were positive clones and the positivity rate of recombinant phage was 78.95%.4 strains with high affinity were obtained after sequencing and analysis,named Ab-5,Ab-7,Ab-8 and Ab-30,respectively.2.Study on the anti-MSRV activity of recombinant antibody proteinE.coli strains of antibodies Ab-5,Ab-7,Ab-8 and Ab-30 were constructed by prokaryotic expression,and the antibody protein expression was detected by SDS-PAGE and Western Blot.The anti-MSRV antibody proteins were prepared by nickel column affinity chromatography and vacuum freeze-drying techniques.On this basis,the affinity of Ab-5,Ab-7,Ab-8 and Ab-30 for MSRV were analyzed by ELISA,and the anti-MSRV activity of 4 antibodies were evaluated at the cellular level in combination with viral infection and antibody treatment.The results showed that Ab-5,Ab-7,Ab-8 and Ab-30recombinant protein were 18 k Da,45 k Da,18 k Da and 18 k Da,respectively.At the same molar concentrations,Ab-7 antibody protein showed higher affinity to MSRV than Ab-5,Ab-8 and Ab-30.There was no significant toxicity to GCO cells at concentrations below300μg/m L of antibody Ab-7.The survival rate of GCO cells was significantly increased to64.87%when the antibody concentration was 150μg/m L compared to the MSRV-treated group.GCO cell survival rates were 70.67%,71.68%and 83.40%at antibody concentrations of 200,250 and 300μg/m L,respectively,and MSRV virus titers were significantly reduced.3.Glycoprotein antigen epitope identification and peptide vaccine immunization effect studyThe physicochemical properties,hydrophilicity and hydrophobicity of antigen G2 and antibody Ab-7 protein were analyzed by bioinformatics.The protein 3D structures of G2and Ab-7 were constructed by Alpha Fold2,and the key antigen regions of G2 were identified by analyzing the antigen-antibody interaction sites by molecular docking.On this basis,the identified G2 key antigen epitope ESQEFTTLTSH was coupled with KLH by chemical synthesis to construct a peptide vaccine.Micropterus salmoides was immunized by intraperitoneal injection,and the immunization effect of the peptide vaccine was evaluated by combining ELISA and virus challenge test.The results showed that both antigen G2 and antibody Ab-7 are hydrophilic proteins with a molecular docking energy of-3.0 kcal/mol.2D interaction map showed that the key amino acids for antigen-antibody interactions were concentrated on the G2^10-20 region.When G2^{Se r11} and G2^{Gln 12} were mutated to alanine respectively,the docking binding energy of both antigen G2 mutation and antibody Ab-7 molecules was greater than 0.Compared with the control group,the serum antibody potency level of Micropterus salmoides was increased significa ntly（p<0.05）after immunization with the peptide vaccine,and the serum antibody potency was1:25600 at 21 d.The results of the MSRV challenge test showed that the relative percentage survival of the peptide vaccine was 62.07%.In summary,this study constructed an anti-MSRV antibody library based on PDT and screened to obtain high-affinity antibodies with anti-MSRV activity.The short peptide ESQEFTTLTSH obtained by bioinformatics analysis is a key epitope for the antigen action of glycoprotein.The KLH-coupled peptide vaccine effectively induced a specific immune response in Micropterus salmoides.The results of this study provided a theoretical basis for the identification of antigen epitopes and vaccine development for aquatic animals.