| Largemouth bass ranavirus(LMBV)is the pathogen of the‘rot disease’of largemouth bass,with a mortality rate of more than 60%,which causes huge economic losses to farmers.At present,there is no effective treatment for the disease,at the same time,drug treatment will bring drug residues,environmental pollution,drug resistance and food safety problems.Immune prevention and control is a feasible scheme,but it is difficult to achieve the injection and anal immunization of fish in water,while immersion immunization has the problem of vaccine degradation,so it is crucial to find a new vaccine.In this paper,the immunoglobulin was extracted from the serum of largemouth bass by saturated ammonium sulfate salting-out method and affinity column chromatography,and BALB/c mice were immunized by conventional methods.The spleen cells were fused with mouse myeloma Sp2/0 cells,and the monoclonal antibody was prepared by hybridoma cell screening and monoclonal cell culture.The titer,sensitivity and specificity of the antibody were determined by enzyme-linked immunosorbent assay.Two hybridoma cell lines A9E7 and C9B9 with monoclonal antibodies against immunoglobulin of largemouth bass were obtained.The ascites antibody titers of A9E7 and C9B9 were 2.19×10~6 and 7.29×10~5,respectively.The sensitivity of A9E7 and C9B9 was 20 ng and 10 ng,respectively.The results of cross-reaction test showed that A9E7 and C9B9 could specifically bind to the immunoglobulin of largemouth bass.A9E7 could cross-react with the serum of seven-star bass,while C9B9 could cross-react with the serum of mandarin fish.There was no cross-react between A9E7 and C9B9 with the serum immunoglobulins of crucian carp,grass carp,blunt snout bream,alburn,mandarin fish,catfish,light lip fish,eel and loach.The largemouth bass were immunized with formaldehyde-inactivated frog siphon virus vaccine,and the high concentration group(≥2.6×10~8/m L TCID50),low concentration group(≥2.6×10~7/m L TCID50)and control group were set respectively.The challenge dose was 0.2 ml/tail.The serum was collected at different time points after immunization,and the antibody titer was determined by using the prepared monoclonal antibody.The results showed that the antibody level in the serum increased significantly after 21 days of immunization,and showed a downward trend after reaching the peak at 35 days,and the antibody titer reached 1:1280.Secondly,the MCPD truncated protein was integrated into the recombinant expression vector p W317 of food-grade Pichia pastoris to construct the recombinant plasmid p W317-mcpd.The recombinant plasmid was linearized by Sal I digestion and transformed into Pichia pastoris GS115 by electroporation.The positive clone GS115-p W317-mcpd was screened by PCR.The transformants were inoculated in BMMY medium and induced with methanol.The induction results were analyzed by SDS-PAGE and Western blotting.The recombinant yeast expressing MCPD protein of the main capsid truncated body of bass frog iridovirus was successfully constructed.The immune effect of recombinant yeast was evaluated by oral immunization with mixture.The experimental groups were set up as 1.0×10~9CFU/g(GS115-MCP)vaccine group,1.0×10~8CFU/g(GS115-MCP)vaccine group,PBS control group,PBS challenge group,and challenge test group after 2 weeks of immunization.The immune protection rate was measured.RT-PCR was used to determine the effects of oral recombinant Pichia pastoris vaccine on the expression of immune-related genes IL-1β,IL-8,MHC I,TNF-αand IFN I in largemouth bass.At the same time,histopathological analysis was performed on liver,spleen,kidney and other tissues in each immune group.The results of challenge protection showed that the immune protection rate after immunization with recombinant bacteria at the concentration of1.0×10~9CFU/g was 60%.Both GS115 empty bacteria group and low concentration recombinant bacteria group showed high mortality after challenge and the mortality rate reached 100%after 10 days of challenge.The results of real-time quantitative PCR showed that the expression of immune-related genes IL-1β,IL-8,MHC I,TNF-αand IFN I in the kidneys of the two groups increased after oral immunization with recombinant Pichia pastoris vaccine,and the expression in the oral immunization1.0×10~9CFU/g(GS115-MCP)group was slightly higher than that in the 1.0×10~8CFU/g(GS115-MCP)group.There was no significant change in the empty vector(GS115)group and the blank control(PBS)group.There was a significant difference between the immune group and the empty vector and the blank control group(P<0.05).Histopathological results showed that the body surface color of the largemouth bass in the non-immunized group was darker than that of the healthy fish,and the pathological anatomy showed expansion and congestion of liver blood vessels.Spleen enlargement;There was a small amount of congestion around the kidney tissue and intestinal food accumulation was not timely digested.After oral administration of recombinant yeast,the damage degree of liver,spleen and kidney in the oral group was significantly improved.The results of indirect enzyme-linked immunosorbent assay showed that the Ig M antibody level in the serum of largemouth bass in the surface oral immunorecombinant strain GS115-MCP group showed an overall trend of increasing first and then stabilizing,and the Ig M antibody level in the109 concentration group was higher than that in the 108 concentration group,which was significantly higher than that in the empty vector GS115 group and PBS control group(P<0.05),and reached the peaknd day(OD490:1.98;OD490:1.65).In this paper,we successfully prepared monoclonal antibodies with strong specificity and high sensitivity of largemouth bass immunoglobulin,and constructed recombinant yeast expressing MCPD protein,which has good immune protection for largemouth bass frog rainbow virus. |
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