| Grass carp reovirus（GCRV）is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp（Ctenopharyngodon idella）.However,management of GCRV infection remains a challenge.To propose the effective prevention or therapeutic strategy for hemorrhagic disease,a series of measures have been developed.Vaccine was applied as the main method to prevent GCRV.DNA vaccine has many advantages such as easy preparation,high safety and effective immune response.However,as an exogenous substance,the DNA vaccine is easy to be degraded and eliminated in tissue resulting in it can not long-term perform the immune function.In this study,a novel vaccine based on Escherichia coli DH5αbacterial ghost,delivering a major capsid protein gene（vp7）of grass carp reovirus encoded DNA vaccine was developed and this ghost-based DNA vaccine was used to evaluate its effects on the prevention of grass carp hemorrhage.Bacterial surface display vaccine has become an increasing strategy for vaccine development because of its potential advantages in immune response.In this study,a novel vaccine was developed based on surface displaying a major capsid protein（VP7）of GCRV on Escherichia coli BL21（DE3）using the surface display technology,further,the outer membrane protein A and major adhesin protein from Aeromonas hydrophila were co-displayed with VP7 protein on the surface of E.coli BL21（DE3）aiming to develop efficient vaccine against GCRV infection by bath administration approach,and the characteristics of the co-displaying vaccine by bath administration approach and its mechanism were studied.The results obtained in this work were as follows:1.Temperature-controlled lysis plasmid（pCAT-lysisE/SNA/Smap29）was constructed by molecular biology technique.E.coli DH5αghosts（DH5α-BG）were produced successfully by induction of temperature up shift from 28°C to 42°C.The lysis kinetics showed that the bacteria lysis efficiency reached 99%after 4 h by inducing plasmid expression.Scanning electron microscopy observation proved that most lysed bacteria appeared lysis holes structure,which mainly distributed in the middle of the bacterial cell or at the polar sites.DH5α-BG were loaded with pcDNA-vp7 by diffusion of plasmid DNA through the lysis holes into the ghosts to obtain the DH5α-BG/pcDNA-vp7 vaccine.Intramuscular immunization for grass carp（1.8-2.0 g）showed:under the same immunizing dose,the antibody levels were significantly enhanced（P<0.05）,acid phosphatase（ACP）activity,alkaline phosphatase（AKP）activity,superoxide dismutase（SOD）activity and serum total antioxidant capacity（T-AOC）were significantly enhanced（P<0.05）,the expression of immune-related genes（TNF-α,IL-1β,IgM,MHCI and IgD）were significantly enhanced（P<0.05）in fish immunized with DH5α-BG/pcDNA-vp7 vaccine.After challenged with GCRV at 21 days post immunization,therelativepercentagesurvival（RPS）reachedto90%in DH5α-BG/pcDNA-vp7 group（5μg for each grass carp）,which was much higher than that in naked pcDNA-vp7（RPS=42.22%）（P<0.01）.Above results indicated that ghost-based vp7DNA vaccine could produce efficient immune protection for grass carp in low vaccine dose.2.Recombinant pET28a-InpN/vp7 plasmid,which contained the N-terminal unique domain of ice-nucleation protein（InpN）and GCRV outer capsid protein vp7 gene,was constructed by molecular biology technique.When the recombinant plasmid was induced expression in E.coli BL21（DE3）,western blot results showed that InpN/vp7 protein were successfully expressed.The analysis of cell fractionation showed that the InpN/vp7 protein accurately located on the outer cell membrane surface and the specific fluorescence signal of the bacterial surface could be observed under fluorescence microscopy The grass carp（2.7-3.0 g）were immunized by surface displaying VP7 protein vaccine（BL21/InpN/vp7）via intraperitoneal injection and bath immunization.The results showed that the specific VP7antibody levels,ACP activity,AKP activity,T-AOC activity and the expression of immune-related genes（TNF-α,IL-1β,MHCI and IgM）were strongly increased（VP7 dose ranged from 20-10μg for grass carp,P<0.01）from 7 to 21 days post injection inoculation in a dose dependent manner.However,BL21/InpN/vp7 vaccine could not effectively induce the increase of above immune parameters post bath immunization for grass carp.After challenged with GCRV at 21 days post immunization,the relative percentage survival in vaccine injection immunization group at the highest dose（20μg VP7 protein）reached to 88.89%.The relative percentage survival in vaccine bath immunization group at the highest concentration(20 mg L-1VP7 protein)only reached to 18.89%.These results indicated that BL21/InpN/vp7vaccine could induce strong immune responses and protect grass carp from GCRV infection by injection immunization.3.The outer membrane protein A gene（OmpA）and major adhesin protein gene（Mah）from Aeromonas hydrophila were inserted into the pET28a-InpN/vp7 plasmid to construct the recombinant pET28a-InpN/vp7-OmpA and pET28a-InpN/vp7-Mah plasmids respectively.TheE.coliBL21（DE3）surfaceco-displayingvp7-OmpAproteinvaccine（BL21/InpN/vp7-OmpA）and vp7-Mah protein vaccine（BL21/InpN/vp7-Mah）were prepared after inducing the expression of recombinant plamids by IPTG.The grass carps were immunized by different surface co-displaying vaccines via intraperitoneal injection and bath immunization.The results of bath immunization showed that the specific VP7 antibody levels,ACP activity,AKP activity,T-AOC activity and the expression of immune-related genes（TNF-α,IL-1β,MHCI and IgM）were significantly increased in BL21/InpN/vp7-Mah vaccine group(VP7 concentration ranged from 20-10 mg L-1 VP7 protein,P<0.01)from 7 to 21days in a dose dependent manner.BL21/InpN/vp7-OmpA vaccine could not effectively induce the increase of above immune parameters post bath immunization for grass carp.The results of injection immunization showed that two kinds of vaccine could significantly induce the increase of above immune parameters from 7 to 21 days post injection immunization for grass carp（VP7 dose ranged from 20-10μg VP7 protein for grass carp,P<0.01）.After challenged with GCRV at 21 days post immunization,the surface co-displaying of vp7-Mah protein vaccine conferred a better protection to grass carp against GCRV infection（relative percentage survival reached to 51.11%）than that in BL21/InpN/vp7-OmpA vaccine group（relative percentage survival only reached to 20%）after bath administration.In addition,the RPS values in surface co-displaying vp7-OmpA protein vaccine group and vp7-Mah protein vaccine group（20μg VP7 protein for grass carp）reached to 82.22%and 91.11%respectively by injection immunization.These results indicated that using the Mah protein to further modify the surface displaying VP7 protein vaccine to obtain the surface co-displaying vp7-Mah protein vaccine,this vaccine could induce strong immune responses and protect grass carp from GCRV infection by bath administration approach.4.Using carp epithelial tumor cell line（EPC）as the cell model,the adhesion and invasin of BL21/InpN/vp7-Mah bacteria to EPC cells were studied by cell lysis counting.The results showed that surface displaying Mah protein can significantly improve the adhesion and invasion of E.coli BL21（DE3）to EPC cells and envelope-mediated endocytosis play a major role in the process of BL21/InpN/vp7-Mah bacteria internalization into EPC cells.The relationship between BL21/InpN/vp7-Mah internalization and the change of EPC cells microfilamentd was studied by flow cytometry and fluorescence imaging techniques.The results showed that the internalization of BL21/InpN/vp7-Mah could cause the microfilament rearrangement and polymerization of EPC cells.Meanwhile,pretreatment of EPC cells with microfilament polymerization inhibitor could significantly inhibit the internalization of BL21/InpN/vp7-Mah.These results indicated that the internalization of BL21/InpN/vp7-Mah depended on the rearrangement and polymerization of EPC cells microfilaments.Further,the effect of BL21/InpN/vp7-Mah internalization on phosphorylation of p38 kinase and HSP27 of EPC cells was studied by western blot.The results showed that BL21/InpN/vp7-Mah internalization can promote the phosphorylation of p38 kinase and HSP27.Meanwhile,pretreatment of EPC cells with p38 kinase inhibitor could inhibit the internalization of BL21/InpN/vp7-Mah.These results indicated that activation of the p38-HSP27 pathway played a role in the internalization of BL21/InpN/vp7-Mah.In summary,bacterial ghost as GCRV vp7 DNA vaccine delivery carrier can enhance the retention and expression time of DNA vaccine in the tissue of grass carp,can enhance the production of specific antibody levels and immune response levels in grass carp,can effectively carry vaccine to immune grass carp.E.coli BL21（DE3）surface displaying VP7protein vaccine can effectively immune grass carp by injection immunization.In addition,further displaying Mah protein on the surface of the E.coli BL21（DE3）,the surface co-displaying vp7-Mah protein vaccine can effectively immune grass carp and protect grass carp from GCRV infection by bath administration approach.Overall,these results provided reference and new approach for the development of vaccine in prevention of grass carp hemorrhagic disease. |
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