Evaluation Of The Immune Efficacy Of Escherichia Coli Outer Membrane Vesicles Presenting Porcine Pseudorabies Virus GD Protein

Pseudorabies(PR)is an infectious disease of pigs,which is caused by Pseudorabies virus(PRV).PRV infection can cause fever,encephalomyelitis,itching,respiratory diseases and growth retardation,sow abortion,stillbirth.Causing huge losses to the global pig industry,PRV has a wide range of hosts,with pigs being the only known natural host,and it is also an important epidemic source.PRV is a member of the herpesvirus family and is mainly composed of 11 glycoproteins.Glycoprotein D(gD)is one of the main envelope proteins of viral particles and has high immunogenicity.In recent years,several gD vaccines that induce gD-specific immune responses have been developed to induce specific antibody against PRV infection.Outer membrane vesicles(OMVs)are small spherical lipid nanoparticles derived from the outer membrane of gram-negative bacteria,with a diameter of 20-300 nm,composed of bacterial proteins,lipids,nucleic acids and periplasmic contents.Although OMVs is released by pathogenic or non-pathogenic bacteria,it does not cause disease because it does not replicate itself.The formation of vesicles is affected by temperature,growth environment and bacterial growth stage.Many gram-negative bacteria naturally release OMVs under environmental pressure during growth,which helps to enhance the adaptability of bacteria.Many outer membrane antigens are enriched on the surface of OMVs,which gives them natural adjuvant properties,indicating that genetic engineering can be used to manipulate OMVs to display heterologous antigens from other bacteria,viruses and parasites,indicating that OMVs can be used as a platform for vaccine design.The purpose of this study was to present PRV gD protein by E.coli outer membrane vesicles,and to study its immunoprotective efficacy and explore its potential as a novel PRV subunit vaccine.Firstly,the recombinant E.coli expressing enhanced green fluorescent protein EGFP was successfully constructed.Using E.coli DH5α as the expression strain and pBAD18 as the vector,the clyA and eGFP gene fragments were successively ligated into the vector.After induction with L-arabinose,the expression of ClyA-EGFP fusion protein in E.coli was confirmed by fluorescence observation and Western blot.The recombinant OMVs expressing EGFP were extracted,and the preparation method of E.coli outer membrane vesicles presenting exogenous proteins was successfully established.Subsequently,EGFP was replaced with gD protein,and the induced bacterial solution was first centrifuged at a low speed to remove the bacteria,and then the supernatant was filtered through a 0.45 μm filter to further remove the bacteria.Subsequently,the supernatant was concentrated by an ultrafiltration concentration tube with a molecular interception of 100 kDa,so that the volume was reduced to 1/4 of the original volume.After ultracentrifugation at 45000 rpm for 2 h,the precipitate was collected as the extracted recombinant outer membrane vesicles.Western blot analysis showed that gD protein was expressed in the outer membrane vesicles.In order to compare the immune efficacy between the OMVs-gD and the prokaryotic expressed gD protein,the gD gene deleted the the signal peptide sequence was cloned into the pET28a prokaryotic expression vector and transformed into E.coli BL21(DE3)competent cells for induced expression.After SDS-PAGE and Western blot identification,the recombinant protein gD-His was successfully obtained.The recombinant protein was purified by nickel column and mixed with Freund ’s adjuvant as the immunogen for later animal experiments.The mice were immunized with OMVs-gD,gD prokaryotic-expressed protein and commercial Bartha K61 vaccine.The protection rate,serum IgG titer,neutralizing antibody titer,viral load and pathological changes were evaluated.The results showed that mice immunized with OMVs-gD could produce higher titer IgG antibody(1:12800);the protection rates of OMVs-gD and gD protein groups were 66.6%and 33.3%,respectively.The neutralizing antibody titer of OMVs-gD group was 1:64,which was significantly higher than that of other groups.The viral load of brain and lung in challenged mice immunized with OMVs-gD was about 103-104 copies/g,and the viral load of heart was about 102-103 copies/g,which was significantly lower than that of other groups(105-107 copies/g).Histopathological observation showed that the brain tissue of the mice in the challenge control group showed obvious lymphocyte infiltration,lung edema,hemorrhage,liver steatosis,and myocardial fiber granular degeneration,while the tissues of the surviving mice in the OMVs-gD group had no obvious lesions.