Preparation Of Bovine Encephalitis Escherichia Coli Multi-epitope Antigen And Evaluate Its Immune Effect

Objective:To screen out the dominant B cell epitopes of FimD,PilN and PilV p roteins of E.coli S9922 causing encephalitis of bovine origin,optimize the sequence of the selected dominant B cell epitopes in tandem,and construct the prokaryotic expr ession vector p ET32a-FimD+PilN+PilV,And to induce expression and purification.Af ter the animal is immunized with the recombinant protein,evaluation of immune prote ction effect.Methods:Bioinformatics analysis tools Prot Param,TMHMM,Net Phos,DNAstar software,SOPMA,SWISS-MODEL,Bepi Pred,Immunomedicine Group,etc.were used to prediction the physical and chemical properties,transmembrane zones,signal peptides,phosphorylation sites,secondary structure,tertiary structure and B cell epitopes of FimD,PilN,and PilV proteins,and made comprehensive analysis to finally obtain the dominant B cell epitopes.Concatenated the dominant B cell epitopes of FimD,PilN,and PilV proteins of E.coli S9922 that cause encephalitis of bovine origin,and optimized the sequence.Th e prokaryotic expression vector p ET32a-FimD+PilN+PilV was constructed and transfor med into BL21(DE3)Escherichia coli for expression.PCR technology and double enzy me digestion technology were used for identification.The transformed BL21(DE3)Esc herichia coli was added with IPTG,and the expression was induced in a bacterial sha king incubator at 37?and 180 rpm.After SDS-PAGE electrophoresis,the expression form of the recombinant protein was analyzed,and the recombinant protein was purifi ed and detected by Western-blot.Preparation of mixed multi-epitope vaccine of Freund's adjuvant and recombinant protein.After immunizing mice,prepare mouse polyclonal antiserum and use indirect ELISA method to detect antibody titer;determine the LD₅₀of bovine encephalitis E.coli S9922 for the bacteria protection experiment,Record the overall situation within 7 days after the bacteria protection experiment,and measure the amount of bacteria in the liver,brain and spleen.Results:Comprehensive analysis of physical and chemical properties,transmembra ne region,signal peptide,phosphorylation site,secondary structure,tertiary structure an d other parameters of FimD?PilN and PilV protein to obtain the final dominant B cel l epitope,FimD protein:7-18,144-152,307-315,365-373;PilN protein:77-86,138-149,516-524;PilV protein:265-275?312-323?402-415.The prokaryotic expression vecto r p ET32a-FimD+PilN+PilV was successfully constructed,and the recombinant protein was expressed in the form of inclusion bodies.Western-blot results showed that a spe cific band appeared,which was consistent with the expected recombinant protein size;the prepared mouse polyclonal antiserum had a titer as high as 1:204800.Western blot results showed that the serum could specifically react with the recombinant expressed protein.The survival rate of mice in the experimental group was 66%;the amount o f bacteria in the liver,brain and spleen of mice in the control group was significantly higher than that of the experimental group.Conclusion:In this experiment,the FimD,PilN and PilV proteins of E.coli S9922caused by bovine encephalitis were comprehensively analyzed,and the dominant B cell epitope was finally obtained.The prokaryotic expression vector p ET32a-FimD+PilN+PilV was successfully constructed,and the recombinant protein contained After the preparation of the polyepitope vaccine,the immune effect of the vaccine was evaluated.The results showed that the vaccine can improve the immunity of mice against bovine encephalitis E.coli S9922,and it can be used for bovine encephalitis E.coli.To lay the foundation for further research on epitope vaccines.