Immunogenicity And Protection Of EV-D68 VP1 Subunit Vaccine And Peptide Vaccine

Objective: The characteristics of capsid protein VP1 of Enterovirus D68(EV-D68)were analyzed by using bioinformatics,and four B cell epitopes of VP1 protein were screened out.These peptides were synthesized to constructed virus peptide vaccine.To constructed virus subunit vaccine,the recombinant VP1 protein of EV-D68 was expressed by prokaryotic expression system and purified by affinity chromatography.The mice were immunized with the two vaccines respectively,and then the titer of the specific antibody were tested and the neutralizing effect of the antibody were compared by challenge test.Method:The physicochemical properties,structural functions and B cell epitopes of EV-D68 VP1 were analyzed by using bioedit,ExPASY,protparam and other bioinformatics tools.The B cell epitopes were screened,synthesized and coupled with KLH protein.Peptide segments were dissolved by PBS and mixed with aluminum hydroxide adjuvant.The VP1 gene of EV-D68 was amplified by PCR and cloned into the prokaryotic expression vector pET28a(+).The recombinant plasmid pET28a(+)-VP1 was constructed and confirmed by restriction enzyme digestion and sequencing analysis,then transformed into E.coli BL21(DE3).The VP1 protein was induced by IPTG.The expressed protein was purified by affinity chromatography.Then purified protein was concentrated and mixed with aluminum hydroxide adjuvant.Peptide vaccine and subunit vaccine of the virus were immunized to mice respectively.The production of antibody and the level of neutralizing antibody were detected.The protective effects of the two vaccines were compared.Result:The basic physical and chemical properties,structure and function of the VP1 protein of EV-D68 were predicted by bioinformatics analysis.The results showed that the protein was a hydrophilic protein with an isoelectric point of 8.73 and a relative molecular weight of 34.0 KD.It had 35 potential phosphorylation sites,no signal peptide and transmembrane region.The highest proportion conformation of the secondary structure was the irregular volume region.?-helix and ?-sheet were scattered in the protein.VP1 protein had many B cell epitopes and 4 optimal epitopes(39-59 aa,82-102 aa,163-181 aa,275-289aa)were selected.The peptide segments were synthesized,coupled with KLH protein,dissolved in PBS solution and mixed with the aluminum hydroxide adjuvant.Five groups of peptide vaccines(Group 5 was the combined group composed of group 1 and group 2)were successfully constructed.The VP1 gene of EV-D68 was amplified by PCR.The result of restriction enzyme digestion of the recombinant plasmid pET28a(+)-VP1 was consistent with the expectation,and the sequencing analysis result was consistent with GenBank.VP1 protein was successfully induced by IPTG and purified by nickel metal chelate affinity chromatography.We successfully constructed the subunit vaccine.Two kinds of vaccines were used to immunize mice.The production of ev-d68 specific antibody in serum was detected by WB.The antibody has good titer of ev-d68 / fermono and ev-d68 / US / IL / 14-18952.The neutralization of antibody to virus was detected by micro serum neutralization test.The results showed that the antibody had no neutralization effect.Conclusion:Bioinformatics technology had certain help for protein structure analysis and epitope prediction.The peptide vaccine of virus had been successfully constructed.The prokaryotic expression vector was constructed and the VP1 protein was expressed and purified.The two vaccines showed good immunogenicity,but the specific antibodies have no neutralization effect.