Construction Of Recombinant Adeno-Associated Virus Expressing GD And GB Proteins Of Porcine Pseudorabies Virus And Mouse Immunity Test

Pseudorabies virus(PRV)is an infectious pathogen which could cause great economic losses to the pig industry.The g B and g D glycoproteins are important immunogenic proteins for PRV.The g B protein and g D protein ecoding genes of the PRV are the target genes for the development of recombinant vector vaccines of the PRV.Using recombinant adeno-associated virus vector(r AAV)expression systemand PRV clinical variant strain PRV/JXFC/2015 genome,AAV2/9 expressing g B and g D glycoproteins of PRV was constructed by PCR technology.The biological characteristics and immunogenicity of the vaccine strain studied in mice.This work laid a valuable foundation for the development of new PRV vaccines.The main contents are as follows:1.Construction of recombinant adeno-associated virus expressing g B and g D glycoproteins of PRVIn this study,the genome of PRV/JXFC/2015 strain virus was used as a sample to amplify g D and g B genes.The g D and g B gene fragments were cloned into the p AAV plasmid by enzyme digestion,respectively.In order to verify the function of the expression system,we constructed the core plasmid p AAV-PRV-g D-P2A-Mcherry and p AAV-PRV-g B-P2A-EGFP carrying the red fluorescent protein(Mcherry)and green fluorescent protein(EGFP)reporter genes,respectively.The plasmids were verified to be correct by enzyme digestion and DNA sequencing.2.Preparation of recombinant adeno-associated virus expressing g B and g D glycoproteins of PRVThe method of preparing r AAV by transfecting HEK-293T cells with three plasmids has successfully produced recombinant AAV2/9 adeno-associated virus expressing g B and g D glycoproteins of PRV.The batch numbers are r AAV2/9-PRV-g B-P2A-EGFP-Z20200103 and r AAV2/9-PRV-g D-P2A-Mcherry-Z20200103.respectively,with a titer of2.5×10¹²vg/m L and 5.00×10¹²vg/m L.3.Immunity test of recombinant adeno-associated virus expressing PRV g B and g D glycoproteins in C57 miceThe mice were immunized by injecting the right lower limb tibialis anterior muscle injection with r AAV2/9-PRV-g B-P2A-EGFP and r AAV2/9-PRV-g D-P2A-Mcherry.In the experimental group,five C57 mice were immunized with each vaccine strain at an immune dose of 5×10¹²vg/kg.The three experimental groups were:group 1(r AAV2/9-PRV-g B-P2A-EGFP),group 2(r AAV2/9-PRV-g D-P2A-Mcherry),group 3(r AAV2/9-PRV-g B-P2A-EGFP and r AAV2/9-PRV-g D-P2A-Mcherry mixed at the same dose).In the commercial vaccine positive control group,five C57 mice were immunized at a dose of 10?L/g.In the negative control group,five C57 mice were immunized with PBS.Then,serum samples were collected at 2,12,16,20,24 and 28th week after immunization.Serum neutralization titers were determined with PRV/JXFC/2015 strain.The serum samples collected on the 28th week after immunization were cross-neutralized by PRV/JXFC/2015 strain and CH-18 strain.The mouse serum neutralizing antibody test was performed.The negative control PBS group and the r AAV2/9-PRV-g B-P2A-EGFP group at all time points,the neutralizing antibody level of the mixed serum of 5 mice were 0;of which the r AAV2/9-PRV-g B-P2A-EGFP and r AAV2/9-PRV-g D-P2A-Mcherry had the same dose of the mixed serum of the mixed injection group,the neutralizing antibody level were 1:8,1:19.95,1:22.9,1:5.6,1:5.01 and 1:31.6,respectively;the neutralizing antibody level of the mixed serum of the positive control commercial vaccine group were 1:16,1:16,1:16,1:16,1:16 and 1:22.38,respectively;the level of neutralizing antibody in the mixed serum of the r AAV2/9-PRV-g D-P2A-Mcherry group were 1:5,1:64,1:32,1:44,1:39.8and 1:56.2,respectively.The cross-neutralization test against CH-18 strain of 28th week mixed serum showed that the neutralizing antibody level of the mixed serum of r AAV2/9-PRV-g B-P2A-EGFP group was 0;the level of neutralizing antibody in the mixed serum of the r AAV2/9-PRV-g D-P2A-Mcherry group was?1:256;and the level of neutralizing antibody in the mixed serum of the r AAV2/9-PRV-g B-P2A-EGFP and r AAV2/9-PRV-g D-P2A-Mcherry group was 1:89.125,The neutralizing antibody level of the mixed serum of the positive control commercial vaccine group was 1:42,the level of neutralizing antibody of the mixed serum of the negative control PBS group was 0.Further determination of IFN-?and IL-4 cytokines in the mixed serum of 5 mice at each time point in each group,the result showed that the highest levels of IFN-?and IL-4were 1973.65 pg/m L and 161.01 pg/m L in the mixed mice of the r AAV2/9-PRV-g D-P2A-Mcherry group,while the highest IFN-?was 1959 pg/m L and the highest IL-4 level was 103.89 pg/m L in the mixed mice of commercial vaccine group.In conclusion,the r AAV2/9-PRV-g D-P2A-Mcherry group had a significant effect in the trial.After a single injection of mice,it stimulated very obvious and long-lasting humoral and cellular immune responses in the mice,reaching a longer Immune effectiveness protection cycle and higher continuous protection effectiveness.