Analysis Of Immune Response Characteristics Of Multi-epitope Vaccines Of Brucella Melitensis Omp10,Omp25,Omp31 And BtpB

Objective: To study the application value of multi-epitope vaccine of Brucella melitensis Omp10,Omp25,Omp31 and Btp B,the physicochemical properties,stability,antigenicity and immune response characteristics of the multi-epitope vaccine were studied.Methods: 1)To explore the general characteristics of Xinjiang brucellosis.2)According to the principle of reverse vaccinology,the proteins with high antigenicity,high stability and high hydrophilicity were selected as target antigens.3)IEDB and Net CTLpan1.1 server were used to predict cytotoxic T lymphocyte(CTL)epitopes,IEDB and Net MHC-IIpan-4.0 were used to predict helper T lymphocyte(HTL)epitopes,BCPREDS were used to predict linear B cell epitopes,and Ellipro was used to predict conformational B cell epitopes.4)Selecting AAY linkers to connect CTL epitopes,GPGPG linkers to connect HTL epitopes,KK linkers to connect B cell epitopes,adding PADRE sequence and human β-defensin-3 at N terminal to increase immunogenicity,finally completing the construction of multi-epitope vaccine,predicting its physical and chemical properties,antigenicity,solubility and tertiary structure respectively,conducting molecular docking with TOLL-like receptor 4,using C-Imm Sim to simulate the immune response after three doses of vaccine injection,selecting p ET-28a(+)as carrier,applying Snapgene to construct plasmid and carrying out simulated agarose gel electrophoresis experiments.5)To verify the immune response characteristics of multi-epitope vaccine by ELISA and ELISPOT.RESULTS: 1)Male patients in Xinjiang’s diseased diseases are higher than female patients;the age range of patients ranges from 0-81 years old,which is good at 50-59 years old;spring is the high incidence of the diseased disease;Tacheng is the high incidence of Xinjiang’s diseased disease;farmers are the main population of infection;cattle and sheep breeding and contact are the main epidemiological characteristics of infection disease.2)After analysis,the target antigens of Brucella melitensis Omp10,Omp25,Omp31,and Btp B were selected.3)After screening,13 CTL epitopes,17 HTL epitopes,9 Linear B cell epitopes and 2 conformational B cell epitopes were selected.4)The constructed multi-epitope vaccine contained 806 amino acids with atomic weight of 86,249.70 KD,instability index of 20.79,average hydrophilicity of-0.459,antigenicity of 0.975,solubility of 0.99,tertiary structure showed that the vaccine contained multiple β-folds and random coiling,molecular docking score of-328.00 and total residue binding energy of Molecular dynamics simulation of-734.058 k J/mol.The results of immunomodulation showed that the vaccine could induce the immune response in the host with the increase of Ig M + Ig G,IFN-γ,TGF-β,IL-10 and IL-18.The constructed protein gene of the vaccine was 2418 bp,and the recombinant plasmid was7753 bp.5)The levels of Ig G1 and Ig G2 a in MEV group were significantly higher than those in control group,and IFN-γ levels in MEV group were significantly higher than those in control group.CONCLUSION: In this study,we studied the spatial,temporal and population distribution of Xinjiang brucellosis,and designed a multi-epitope vaccine based on Omp10,Omp25,Omp31,and Btp B,it is a good choice for vaccine design.This study provides a theoretical basis and a new immunological idea for the prevention of brucellosis.