| Grass carp hemorrhagic disease(GCHD)is one of the most threatening diseases to grass carp,causing significant economic losses to the grass carp breeding industry.At present,Grass carp reovirus type Ⅱ(GCRV-Ⅱ)is the most important pathogen causing GCHD outbreaks.Vaccine immunization is an important method to prevent GCHD.Among them,the immersion immunization method has the advantages of saving time and effort and convenient for commercial promotion.However,the vaccine immunizes the fish through the immersion method and it is difficult to break through the biological barrier to exert immunity.This study used genetic engineering technology to screen the epitope of GCRV-Ⅱ VP4 protein,and used carbon nanotubes as a carrier and mannose as a targeting factor to construct a carbon nanotube-containing vaccine system with targeted immune function,and evaluate the vaccine’s effectiveness.Immune protection effect,and optimize the immune program,determine the immune protection program that can be industrialized.The main content includes the following aspects:1.Screening of dominant epitopes of VP4 proteinAccording to the vp4 gene sequence(1953 bp)of the representative strain of GCRV-Ⅱ,it was truncated into 4 segments,and constructed containing p ET32a-vp4,p ET32a-vp4-1,p ET32a-vp4-2,p ET32a-vp4-3 and p ET32a-vp4-4(vp4-1 fragment size is 534 bp,vp4-2,vp4-3 and vp4-4 fragment size is 537 bp)genetically engineered strain of prokaryotic expression plasmid.After verification by double enzyme digestion and sequencing,Escherichia coli BL21(DE3)was introduced to induce protein expression,and the protein expression and activity were detected by polyacrylamide gel electrophoresis and western blotting.Finally,the enzyme-linked immunosorbent assay was used to screen the protein’s dominant antigen epitopes.The results showed that the OD450 value of VP4-3 was 1.7609 significantly higher than that of other proteins,and it could be used as a candidate vaccine antigen for preventing GCHD.2.Preparation of targeted carbon nanotube vaccine systemUsing SWCNTs as a carrier and Mannose as a targeting factor to construct a carbon nanotube-carrying subunit vaccine system(SWCNTs-VP4 and SWCNTs-VP4-3,in which VP4 and VP4-3 antigen proteins accounted for approximately 52.23% and 53.19%respectively),the target Subunit vaccine system containing directional carbon nanotubes(SWCNTs-M-VP4-3,of which VP4-3 antigen protein accounts for about 45.27%).The grass carp was immunized with a targeted carbon nanotube-containing vaccine system of different concentrations(5,10,20,40,and 80 mg/L)for 6 hours.After 28 days of immunization,the safety test results showed that the grass carp body activity was normal.The mortality rate was below 10%;compared with the control group,there were no significant differences in lipase and amylase activity,growth performance,and muscle growth-related gene expression(Myf-5,Myo D1,and Myo G).This chapter successfully constructed a targeted carbon nanotube vaccine system for the prevention and treatment of grass carp hemorrhagic disease,which is relatively safe for grass carp within 80 mg/L.3.Immune effect research of targeted carbon nanotube vaccine systemWith VP4(5.0 mg/L),SWCNTs-VP4(1.0 mg/L,2.5 mg/L and 5.0 mg/L),SWCNTs-VP4-3(1.0 mg/L,2.5 mg/L and 5.0 mg/L)Grass carp was immersed and immunized with different vaccine systems(1.0 mg/L,2.5 mg/L and 5.0 mg/L)from SWCNTs-M-VP4-3(1.0 mg/L,2.5 mg/L,and 5.0 mg/L)for 6 hours,by measuring immune-related physiological indicators,serum antibody titers,and immune-related gene expression And the relative immune protection rate to evaluate the immune protection effect of the vaccine system.The results showed that,compared with other groups,the expression levels of immune-related genes,immune-related physiological indicators and serum antibody titer levels in the SWCNTs-M-VP4-3 group were significantly increased.Virus challenge results showed that the highest concentration group of SWCNTs-M-VP4-3had the highest immune protection rate of 60.95%,while the highest concentration group of SWCNTs-VP4-3,SWCNTs-VP4 and VP4 groups had a relative immune protection rate of51.43%,42.86% and 33.33%,respectively.The targeted carbon nanotube vaccine system made of dominant antigens can effectively enhance the immune protection effect of grass carp against GCHD through immersion immunity.4.Research on the effect of vaccine applicationThe different immunization parameters of the SWCNTs-M-VP4-3 vaccine system were optimized through a complete experiment,that is,different immunization concentrations(5,10,and 20 mg/L),immunization time(6,12,and 24 h)and different fish densities(15,30,60 tails/L),the grass carp was immunized by soaking method to evaluate the immune protection effect of the vaccine.According to the results of the challenge test,when the immunization time is 12 hours,the immunization concentration is 10 mg/L,and the fish density is 15 tails/L,the highest immune protection rate is 88.33%.The results of immune-related physiological indicators,serum antibody titer and immune-related gene expression levels also reached the same conclusion.According to the above-mentioned immunization plan,the grass carp in a fish breeding farm was immersed and immunized.Virus challenge results showed that the average survival rate of the control group was11.83%,the average survival rate of the grass carp treatment group was 84.67%,and the relative immune protection rate was 82.61%.Immunization of grass carp through this program can significantly improve the survival rate of fry.In summary,this thesis successfully screened the dominant epitope of GCRV-Ⅱ VP4 protein and prepared a carbon nanotube-borne vaccine system with targeted functions,which can achieve high-efficiency protection of grass carp and determine the most suitable immunity through optimization of immune procedures The plan is that the vaccine is immersed for 12 hours,the immunization concentration is 10 mg/L,and the immersion density is 30 g/L.Good results have been achieved in the industrial application of fish seed farms,which is of great practical significance.This study will provide new ideas for other aquatic animal nano-borne vaccine systems,and can provide a certain reference for the promotion of other aquaculture vaccines. |
