| Fusobacterium necrophorum is a gram-negative anaerobic pleomorphic bacterium that mainly causes liver abscess,hoof rot,and calf diphtheria,and plays an important role in the development of dairy cow mastitis and endometritis.The adhesion of Fusobacterium necrophorum bovis to host cells is a key step for its invasion of the body and pathogenicity.Therefore,it is of great significance to clarify the main sites of 43 K OMP’s adhesion and to screen the corresponding epitopes.This experiment starts from the adhesion function of Fusobacterium necrophorum bovis 43 K OMP and host cell,screens the adhesion function area of 43 K OMP;prepares its monoclonal antibody,and preliminarily determines the adhesion site of the monoclonal antibody;combines bioinformatics analysis technology to screen And verify the epitope of the 43 K OMP of Fusobacterium necrophorum bovis.The experiment first used the prokaryotic expression system to recombine and express four overlapping truncated fragments of the 43 K OMP gene of Fusobacterium necrophorum bovis.The expressed protein was purified and prepared rabbit polyclonal antibodies.The polyclonal antibodies were prepared by the caprylic acid-saturated ammonium sulfate method.The crude and purified polyclonal antibodies were tested for adhesion inhibition with BEND and MACT cells respectively,and the adhesion inhibition effects of the 4 polyclonal antibodies were evaluated by the Gram staining method and plate counting method to preliminarily determine the adhesion of 43 K OMP functional area;then prepare the monoclonal antibody of 43 K OMP adhesion functional area,ELISA to detect the monoclonal antibody titer,antibody subclass detection kit to determine its subclass,Western blot to identify the monoclonal antibody,and preliminary screening of the monoclonal antibody recognize epitopes;finally use bioinformatics technology to analyze the physical and chemical properties,transmembrane region,signal peptide,phosphorylation site,secondary structure,tertiary structure,hydrophilicity,accessibility,flexibility,and B cells of 43 K OMP Epitope and other parameters,comprehensive bioinformatics analysis results to select the antigen epitope located in the adhesion functional area,and finally the selected predicted epitope peptide is synthesized,and the reactivity of the synthetic peptide is detected by indirect ELISA and indirect immunofluorescence method.The adhesion of synthetic peptides to BEND cells and MACT cells,and finally a competitive inhibition test with synthetic peptides to verify its inhibitory effect on the adhesion of Fusobacterium necrophorum.The results are as follows:(1)The truncated fragment of the 43 K OMP gene of Fusobacterium necrophorum was successfully expressed and purified,and a polyclonal antibody was prepared.Through the adhesion inhibition test of the polyclonal antibody with BEND and MACT cells,two adhesion functional regions were successfully screened,respectively.They are 19～108aa and 283～377aa;(2)A monoclonal antibody located in the adhesion functional area was successfully prepared,named 1G7,the titer of the supernatant was 1:3 200,and the titer of ascites was 1:104.Preliminary identification of its recognition site is 283～306aa;(3)Through bioinformatics analysis technology,it is analyzed that 43 K OMP has multiple phosphorylation sites,no transmembrane region,and signal peptide region at 1～20aa.B cell epitopes,10 conformational B cell epitopes were predicted,and 4 linear B cell epitopes located in the adhesion functional area were selected and synthesized.The specific amino acid sequence is T1(49～63aa): VVQAPAKWKPNGSVG;T2(72～89aa): VENKGKKATEENARKGWA;T3(386～302aa): ETFWAWDKKDASMEEWP;T4(348～365aa): GEYVNRENNKSTARYWRW.It is verified that it has good immune activity,good adhesion to BEND cells and MACT cells,and can compete to inhibit the adhesion of Fusobacterium necrophorum to BEND cells and MACT cells.In this experiment,43 K OMP adhesion functional areas were initially screened,ranging from 19～108aa and 283～377aa.A 43 K OMP monoclonal antibody against Fusobacterium necrophorum bovis was successfully prepared and named 1G7,and its recognition epitope was283～306 aa.It is predicted that the 43 K OMP of Fusobacterium necrophorum bovis has 10 conformational B cell epitopes;4 epitope peptides located in the adhesion functional area,T1:VVQAPAKWKPNGSVG;T2: VENKGKKATEENARKGWA;T3:ETFWAWDKKDASMEEWP;T4: GEYVNRENNKSTARYWRW has been verified to have good immune activity,can adhere to BEND cells and MACT cells,and can compete to inhibit the adhesion of Fusobacterium necrophorum bovis to BEND cells and MACT cells.It provides a theoretical basis for further research on the pathogenic mechanism of Fusobacterium necrophorum bovis and the development and design of antibacterial drugs. |
