Construction Of Self-Assembled Nano Vaccine Of Carp Spring Viremia Virus And Its Immune Effect

Spring virus of carp(SVC)is a serious viral disease in fish,and vaccination is an important measure to prevent and control SVC.Immersion vaccine has become an important development direction of fish vaccine research,because it is more suitable for the actual situation in the aquaculture process.With the extensive development of nanomaterials research,self-assembled peptides have become a research hotspot for new fish immersion vaccines due to its unique physical and chemical properties and high loading capacity.In this study,the selfassembled peptide Q11(QQKFQFQFEQQ)was used as the vaccine carrier,and SVCV dominant epitope SVG(NAGYFSRDVRGE)was used as the antigen,and the SVCV selfassembled nanovaccine system SVG-Q11(NAGYFSRDVRGESGSGQQKFQFQFEQQ)was prepared and constructed by solid-phase synthesis technology.Combining transmission electron microscope(TEM),circular dichroism(CD),dynamic light scattering(DLS)and other methods to identify the structural morphology and characteristics of SVG-Q11.And evaluation of the safety of the vaccine SVG-Q11 at the cellular and individual levels.On this basis,the preventive effect of the vaccine system on SVC was comprehensively evaluated through immersion immunization and immune challenge experiments,and the vaccine immunopotentiation mechanism was explored.The content of the thesis mainly includes the following aspects:1.Construction of self-assembled nanovaccine systemThe self-assembled peptide Q11 with the strongest penetration effect was screened by flow cytometry and in vivo imaging technology,and the SVCV dominant antigenic epitope SVG screened in the laboratory was connected to Q11 by using the flexible linker peptide SGSG to construct a self-assembled nanovaccine system SVG-Q11,and carried out solidphase synthesis to obtain Q11,SVG,and SVG-Q11 with molecular weights of 1.53 k Da,1.37 k Da,and 3.13 k Da,respectively.Then use TEM,DLS and CD to analyze the shape,particle size and ellipticity of the three.The results show that Q11 and SVG-Q11 are elongated nanofiber structures,and the SVG structure is in the form of individual particles;DLS results show that the average particle sizes of Q11,SVG and SVG-Q11 are: 67.51 ± 4.88 nm,167.57± 2.17 nm,respectively,182.93 ± 3.52 nm;CD results showed that Q11 and SVG-Q11 had characteristic peaks consistent with the CD spectral distribution of the protein β-sheet secondary structure,and no characteristic peaks were observed in SVG.The above results indicated that the SVG-Q11 self-assembled nanovaccine was successfully constructed.2.Safety evaluation of self-assembled nanovaccine systemThe safety evaluation of Q11,SVG,and SVG-Q11 was carried out at the cell and individual levels,respectively.At the cellular level,the three vaccines were co-incubated with carp macrophages and epithelioma papulosum cyprini(EPC)cells for 24 h,respectively,and the cell viability was detected by MTT method.At the individual level,different concentrations of SVG-Q11 were used to soak carp for 6 h,and then transferred to clean water for 28 days to observe the survival of the fish.After 28 days of immersion treatment,histological sections of the spleen,gills,intestinal tract,kidneys,liver,and other organs of carp in the control group and the highest concentration SVG-Q11 group(40 mg/L)were prepared and observed.The results showed that: at the cell level,the survival rates of carp macrophages and EPC cells in each treatment group were above 90%.There was no significant difference in various growth indicators such as 40 mg/L.At the highest treatment concentration of 40 mg/L,there was no significant difference between the spleen,gills,intestines,kidneys,liver and other tissues and organs of carp and the control group,and no tissue lesions or anomalies were observed.3.The immune effect evolution of self-assembled nanovaccine systemImmunize carp through immersion,and comprehensively evaluate the immune protection effect of the self-assembled nanovaccine system by combining changes in immune related indicators and challenge experiments.The results showed that after the SVG-Q11(20 mg/L)group was used to immunize carps for 6 h,compared with the control group,Q11(20 mg/L)group and SVG(20 mg/L)group,the serum antibody titer level in the SVG-Q11(20 mg/L)group was the highest,and was significantly higher than other groups(P < 0.05);the alkaline phosphatase(AKP),complement C3(Complement C3),superoxide dismutase(SOD)and acid phosphatase(ACP)were significantly higher than other groups(P < 0.05);SVG-Q11(20mg/L)group also had significantly higher expression levels of immune-related genes(TNF-α,IL-1β,Cxcr 1,Cxca,IFNg2 b,Ig M,CD4,and MHC II)than other groups(P < 0.05).After using SVCV to challenge carps immunized in baths,the immune protection rate of the highest concentration group(20 mg/L)of SVG-Q11 vaccine was up to 73%.4.Research on the immune enhancement mechanism of self-assembled nanovaccine systemThe delivery pattern of SVG-Q11 in fish and organs was analyzed by in vivo imaging technology.This was followed by a study of the delivery effects of Q11,SVG,and SVG-Q11 in various tissue and organ systems of fish using tissue section immunofluorescence.And The activation level of antigen-presenting cells in immunized carp was measured through ELISA analysis.The results showed that SVG-Q11 could enter the fish body through immersion and be delivered to systemic tissues and organs including the gills and intestines.At the cellular level,fluorescence assays revealed that the levels of Q11 and SVG-Q11 were significantly higher in EPC cells than in SVG and the control groups(P < 0.05).Tissue fluorescence analysis showed that the uptake of Q11 and SVG-Q11 in the kidney and spleen of carp was higher than that in SVG and the control groups.The results of antigen presentation activating factors showed that the expression levels of CD80/86 and TNF-α in the spleen and kidney of the SVG-Q11 group were significantly higher(P < 0.05)compared to the control,Q11,and SVG groups.In summary,the self-assembled nanovaccine SVG-Q11 constructed in this study could provide efficient immune protection by improving the ability of the vaccine to penetrate biological barriers,increasing the vaccine content in fish after immunization and enhancing the antigen presentation response of the vaccine.This study could provide a theoretical basis for the research of self-assembled nanovaccine in the aquatic industry,and provide an efficient and safe solution for the prevention and control of aquatic animal diseases and the healthy development of aquaculture.