| Vibrio mimicus is a gram-negative bacillus with a short arc-shaped rod shape and no capsule and spores.It is one of the main pathogens of the Vibrio genus.It can infect humans and cause diseases such as otitis media and trauma infection;infect aquatic animals and cause ulcers and ascites disease.It has serious harm to human health and safety and fish breeding industry.Extracellular products(ECPs)refer to the metabolites secreted by pathogenic bacteria to the outside of the cell during the process of growth and reproduction.They can assist pathogens to cause disease together,and can also cause immune response in the host.They can be used as a good Protective antigen.In this paper,the various enzyme activities and pathogenicity of the extracellular products of four strains of Vibrio mimicus were tested,laying a foundation for clarifying the biological characteristics of the extracellular products of the bacteria;further testing and analyzing the protein components of the extracellular products,and immunizing the crucian carp to observe the protection effect.Lay the foundation for the follow-up vaccine research and development of Vibrio mimicus.Firstly,the extracellular products of four strains of Vibrio mimicus were prepared by ultrafiltration and concentration method,and the activities of 7 kinds of extracellular enzymes was detected by the plate punch method,and the influence of physical and chemical factors on the activity of extracellular enzymes of the four strains of Vibrio mimicus was analyzed.The HSY-K0531 strain of Vibrio mimicus with the strongest extracellular enzyme activity was selected.Preliminary analysis of the protein composition of extracellular products by SDS-PAGE,determination of the protein concentration and LC-MSMS method to analyze the types and quantities of proteins in the extracellular products of HSY-K0531 strains.At the same time,determine the half lethal dose of the extracellular products of Vibrio mimicus HSY-K0531 strain to crucian carp.The results showed that the four strains of Vibrio mimicus all possess protease,hemolytic enzyme and DNase,and have a certain range of adaptation to temperature,culture time and p H.The relevant enzyme activity of the Vibrio mimicus HSY-K0531 strain is the strongest,and its protein composition is the most active.In the analysis,a total of 101 proteins were detected,mainly including various protein molecules with enzymatic activity and immunogenicity,which participated in 872 biological processes and 454 molecular functions.At the same time,the median lethal dose of extracellular products of Vibrio mimicus HSY-K0531 strain to crucian carp was 116.1 μg/tail.The extracellular protein of the Vibrio mimicus HSY-K0531 strain and the inactivated vaccine were used to immunize the crucian carp to observe and compare the immunoprotective effects.Three test groups and a control group were the extracellular product + Freund’s adjuvant group.Inactivated vaccine + Freund’s adjuvant group,extracellular product mixed inactivated vaccine + Freund’s adjuvant group and PBS control group were immunized twice,two weeks after the first immunization,the second immunization was carried out,and 5 were collected every other week after the immunization.The serum and liver,spleen,kidney,intestine,and gills of the tail crucian carp were sampled 5 times in total with pre-immunization sampling as a control.Serum is used in ELISA kit to detect immunoglobulin M(Ig M),superoxide dismutase(SOD),alkaline phosphatase(AKP),acid phosphatase(ACP),lectin(Lectin),lysozyme(LZM)Vitality,the activity of complement(C3)and(C4)in the serum.The results showed that the activities of related immune enzymes,immune factors and non-specific immune antibodies in the blood of crucian carp in the three test groups were improved to varying degrees,which enhanced the level of humoral immunity level.At the same time,the results of fluorescence quantitative PCR analysis showed that the expression of the four immune factors TGF-β,TNF-α,IL-1βand IL-10 in the organs of the crucian carp tissues of the three test groups all increased to varying degrees,which enhanced the crucian carp Cellular immunity level.The results of the challenge protection test showed that the protection rate of the extracellular protein group was 60%,the inactivated vaccine group was 55%,and the mixed group was 43%.All three groups can improve the ability of crucian carp against Vibrio mimicus infection,and the extracellular protein group is immune the best results.In summary,this study compared the biological characteristics of four strains of Vibrio mimicus,and selected a strain of Vibrio mimicus HSY-K0531 with better extracellular protease activity,and compared the extracellular protein and inactivation of this strain.The immune protection effect of the vaccine and the research results provide a theoretical reference for the development of the subunit vaccine of the extracellular product of Vibrio mimicus. |
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