Design,Preparation And Evaluation Of Porcine Epidemic Diarrhea Virus Protein Vaccine

Porcine epidemic diarrhea virus(PEDV)can infect pigs of different ages and cause porcine epidemic diarrhea(PED),causing heavy economic losses to the pig industry around the world.There is currently no effective treatment for PEDV,and vaccination is its key prevention and treatment method.This research carried out the design and molecular construction of subunit vaccines,preparation process establishment and immune evaluation for PEDV.At the same time,it also carried out the preliminary exploration of PEDV virus-like particles(VLPs)vaccine.(1)In this study,based on the study of PEDV epitopes,a subunit vaccine with tandem epitopes was designed and a baculovirus expression system was constructed.The COE region(E1),S1D region(E2),C-terminal region(E3)of spike protein and the M3 region(E4)of membrane protein were selected,and the above-mentioned epitopes were connected by linker to form a tandem epitope vaccine(EC).At the same time,the COE area and S1 area,which have been studied more often,are used as control subunit vaccines.The EC,COE,and S1 fragments were inserted into baculovirus vectors respectively.Sf9 cells were transfected with different recombinant baculovirus vectors to obtain the target recombinant baculovirus,and preliminary expression tests were carried out.The results confirmed that each protein can be secreted and expressed in sf9 cells.(2)In this study,the production process of PEDV subunit vaccine was established under the baculovirus expression system.First,the effects of different multiple of infection(MOI)and time of infection(TOI)on the expression levels of each target protein were compared,and the optimal expression conditions were determined.Next,use nickel column affinity chromatography to purify each target protein,and explore the best elution conditions for the target protein.The results confirmed that the expression levels of EC protein and COE protein were higher under the conditions of MOI of 0.5～1 and TOI of 4～5 d,and the expression levels of S1 protein were higher under the conditions of MOI of 1～5 and TOI of 2 d.Most EC proteins can be eluted at 50 mmol/L imidazole,most COE proteins can be eluted at 10 mmol/L imidazole,and most S1 proteins can be eluted at 50 mmol/L and 100 mmol/L imidazole.Under the 8 L expression system,the output of EC,COE and S1 protein reached 0.5 mg/L,1.7 mg/L and 2.8 mg/L,respectively.(3)In this study,the immune effect of different unit vaccines was evaluated on BALB/c mouse.The results confirmed that when aluminum glue was used as an adjuvant,the subunit vaccine EC stimulated mouse to produce higher levels of specific antibody IgG and immune-related cytokines IFN-y and TNF-α.Next,the immune effect of the S1 subunit vaccine under different adjuvants was compared,and the results confirmed that the S1 subunit vaccine combined with ISA 61 VG mineral oil adjuvant and Poly(I:C)immune enhancer stimulated mouse to produce higher levels specific antibody IgG and immune-related cytokines IFN-y and TNF-α.(4)This study carried out the preliminary exploration of PEDV VLPs vaccine.First,three promoters,pSeL120,p10,and actin3,were selected from the promoters used in the baculovirus expression vector systems(BEVS),and the promoters were used to activate the spike(S)protein,membrane(M)protein and envelope(E)protein genes,respectively.expression.Next,the above-mentioned DNA sequence was constructed into the baculovirus genome,and sf9 cells were infected with the prepared recombinant baculovirus to prepare PEDV VLPs.The results prove that S,M and E proteins can be expressed in sf9 cells and form VLPs spontaneously.However,the output of VLPs is low,and efforts need to be made to increase the output.In this study,it was confirmed that the tandem epitope vaccine EC can better stimulate the humoral and cellular immune responses of mouse,and has the potential as a PEDV subunit vaccine.The combination of ISA 61 VG mineral oil adjuvant and Poly(I:C)immune enhancer has a better immune enhancement effect and has the value as a PEDV vaccine adjuvant.This study has certain reference value for the development of PEDV subunit vaccines.