Preparation Of DNA Vaccine Against Nocardia Seriolae In Largemouth Bass,micropterus Salmoides And Evaluation Its Immune Effect

Largemouth bass（Micropterus salmoides）nocardiosis is a highly harmful epidemic disease caused by Nocardia seriolae,which affects the development of largemouth bass culture.Because of the long incubation period and high incidence,it often leads to a large number of sick fish die,causing huge economic losses to largemouth bass farming industry.Currently,the traditional drug treatment of largemouth bass nocardiosis involves a variety of sensitive antibiotics,but the efficacy is very limited,and there are serious environmental pollution,microbial resistance and drug residues.In contrast,vaccines in aquaculture have the advantages of safety,environmental protection and long-term protection,among which DNA vaccines have better immune effect on intracellular bacteria.Based on this,In this study,ATP-binding protein（ABP）,Penicillin-binding protein 2（PBP2）and Serine/threonine protein were obtained from ABC transporter kinase（STPK）,Phospholipase C（PC）and MCE family protein（MFP）are selected by detecting the regenicity of five antigen candidates,including kinase（STPK）,Phospholipase C（PC）and MCE family protein（MFP）,then the candidate antigen with the best immunogenicity was obtained by testing the immunogenicity of the candidate antigen with better reactivity;according to the candidate antigen obtained by screening,the DNA vaccine encoding the antigen was prepared and its immune effect was evaluated.The main results are as follows:1.Screening and immunogenicity detection of dominant candidate antigensAccording to the gene sequences of the five candidate antigens,the engineering strains containing the prokaryotic expression plasmids of pET32a-ABP,pET32a-PBP2,pET32a-STPK,pET32a-PC and pET32a-MFP were constructed,respectively.Escherichia coli BL21（DE3）was introduced after double enzyme digestion and sequencing verification.The expression and activity of the protein were detected by SDS-PAGE and western blotting.According to enzyme-related immunosorption assay,the reactivity of five candidate antigens was tested.The results showed that the OD₄₅₀values of ABP and PBP2 were 0.861 and0.672,which were significantly higher than those of other proteins.Therefore,ABP and PBP2 candidate antigens had good reactivity2.DNA vaccine preparationThe purified ABP and PBP2 were immunized against largemouth bass at 2μg/tail and10μg/tail.The immunogenicity of ABP and PBP2 was evaluated by ELISA,real-time quantitative PCR and Nocardia seriolae infection test.The results showed that ABP（10μg/tail）,ABP（2μg/tail）and PBP2（10μg/tail）treatment groups significantly increased serum antibody levels and related immunoenzyme activity levels,and significantly up-regulated the expression of immune-related genes.The immunized largemouth bass were artificially infected with Nocardia seriolae,the results of ABP（10μg/tail）treatment group were significantly higher than those of PBP2（10μg/tail）treatment group.The results showed that the relative immune protection rates of ABP（10μg/tail）,ABP（2μg/tail）,PBP2（10μg/tail）and PBP2（2μg/tail）groups were 70%,40%,30%and 15%,respectively.To prove that ABP has better immunogenicity,ABP was selected as the candidate antigen.3.Evaluation of immune effect of DNA vaccineDNA vaccine encoding ABP（pcDNA-ABP）and DNA vaccine encoding ABP coated with mannosaccharated chitosan（MCS-pcDNA-ABP）were prepared successfully.The DNA vaccine was injected into the dorsal fin muscle to immunize largemouth bass,the immunoefficacy of the DNA vaccine was evaluated by ELISA,real-time fluorescence quantitative PCR and Nocardia seriolae challenge test.The results showed that compared with the control of pcDNA and MCS-pcDNA,the serum antibody levels and immune-related enzyme activity levels of largemouth bass in pcDNA-ABP and MCS-pcDNA-ABP groups were significantly increased,and the expression of immune-related genes was significantly up-regulated.The relative immune protection rate of MCS-pcDNA-ABP and pcDNA-ABP treatment groups was 80.73%and 69.23%,respectively,after Nocardia seriolae challenged the immunized larguid bass in each group.It is proved that DNA vaccine has protective effect against Nocardia seriolae,and the protective effect of DNA vaccine coated with mannosaccharated chitosan is better.In conclusion,the DNA vaccine coated with mannosaccharated chitosan constructed in this study can produce a significant systemic immune response,so as to have a good preventive effect against Nocardia seriolae.This study not only provides theoretical basis and experimental data for the development of DNA vaccine against Nocardia seriolae infection,but also provides effective strategies for the prevention and control of largemouth bass nocardiosis.