Vibrio vulnificus inactivated vaccine and preparation method and application thereof

文档序号：293971 发布日期：2021-11-26 浏览：15次中文

阅读说明：本技术 一种创伤弧菌灭活疫苗及其制备方法和用途 (Vibrio vulnificus inactivated vaccine and preparation method and application thereof ) 是由张敏顾勤勤王光花郝东方刘红梅于 2021-08-20 设计创作，主要内容包括：本发明提供了一种创伤弧菌灭活疫苗的制备方法,包括：1)将TC38抗菌肽加入到创伤弧菌菌液中,进行灭活处理,得到灭活的创伤弧菌孵育液；2)将所述灭活的创伤弧菌孵育液离心,收集菌体沉淀,得灭活菌体；3)将所述灭活菌体以无菌PBS清洗数次后,分散于无菌PBS中,即得灭活疫苗。本发明还提供了相应的灭活疫苗及其应用。本发明以TC38抗菌肽灭活创伤弧菌得到了一种新型灭活疫苗,该疫苗在大菱鲆中诱发的相对免疫保护率显著高于福尔马林灭活疫苗组,可诱导鱼体产生更高的非特异和特异性免疫应答水平,在水产动物创伤弧菌病害防治中具有良好的应用前景。(The invention provides a preparation method of vibrio vulnificus inactivated vaccine, which comprises the following steps: 1) adding TC38 antibacterial peptide into vibrio vulnificus bacterial liquid, and inactivating to obtain inactivated vibrio vulnificus incubation liquid; 2) centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting thallus precipitates to obtain inactivated thallus; 3) and (3) cleaning the inactivated thallus for a plurality of times by using sterile PBS, and dispersing the inactivated thallus in the sterile PBS to obtain the inactivated vaccine. The invention also provides a corresponding inactivated vaccine and application thereof. The TC38 antibacterial peptide is used for inactivating vibrio vulnificus to obtain a novel inactivated vaccine, the relative immune protection rate of the vaccine induced in turbots is obviously higher than that of a formalin inactivated vaccine group, the fish body can be induced to generate higher non-specific and specific immune response levels, and the vaccine has a good application prospect in preventing and treating vibrio vulnificus diseases of aquatic animals.)

1. A preparation method of Vibrio vulnificus inactivated vaccine is characterized by comprising the following steps:

1) adding TC38 antibacterial peptide into vibrio vulnificus bacterial liquid, and inactivating to obtain inactivated vibrio vulnificus incubation liquid;

2) centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting thallus precipitates to obtain inactivated thallus;

3) and (3) cleaning the inactivated thallus for a plurality of times by using sterile PBS, and dispersing the inactivated thallus in the sterile PBS to obtain the inactivated vaccine.

2. The method of claim 1, further comprising:

4) carrying out sterile inspection on the inactivated thallus;

preferably, the sterility test is achieved by a method comprising the steps of: and diluting the inactivated bacteria with sterile PBS, taking a proper amount of the diluted inactivated bacteria, coating the diluted inactivated bacteria on a solid culture medium, and culturing for 24-48 h at 28 ℃ to confirm whether the Vibrio vulnificus is completely inactivated.

3. The method of claim 1, wherein the vibrio vulnificus solution is prepared by a method comprising the following steps:

resuscitating vibrio vulnificus stored at-80 ℃ to the middle logarithmic growth phase by using a liquid LB culture medium, centrifuging, cleaning, and then suspending in sterile PBS to obtain the vibrio vulnificus suspension; preferably, the concentration of the vibrio vulnificus bacterial liquid obtained after resuspension is 2 × 10⁸CFU/mL。

4. The method according to any one of claims 1 to 3, wherein the TC38 antibacterial peptide is a polypeptide having an amino acid sequence having a homology of not less than 85% with SEQ ID NO. 1.

5. The inactivated vaccine of Vibrio vulnificus prepared by the preparation method according to any one of claims 1 to 4.

6. A medicament for preventing or treating Vibrio vulnificus infection in a marine cultured animal, comprising the Vibrio vulnificus inactivated vaccine of claim 5; preferably, the effective amount is 0.182 mg/strip.

7. Use of the inactivated vaccine of Vibrio vulnificus according to claim 5 in the preparation of a medicament for preventing or treating Vibrio vulnificus infection in a marine cultured animal.

8. The use according to claim 6, wherein the marine farmed animals are selected from one or more of eel, grouper, sturgeon, tilapia, golden pomfret or turbot.

Technical Field

The invention relates to the technical field of animal medicine, in particular to a vibrio vulnificus inactivated vaccine and a preparation method and application thereof.

Background

Vibrio vulnificus, which is a vibrio copropathic of human and marine life, is mainly of three types: biological type I, which is susceptible to ingesting cases and wound infections; organism type II, the pathogenic type responsible for disease in marine farmed animals; and type III organisms capable of infecting humans (Wu Backuwa and Pan Kimura, 2001; Bisharat et al, 1999; Kelly and McCormick, 1981; Tison et al, 1982). The pathogenicity of vibrio vulnificus is a result of the combined action of various pathogenic factors, mainly including hemolysin, MARTX toxin, siderophores, capsular polysaccharide, lipopolysaccharide, metalloprotease and the like (arborvitae regolii, 2018; Li and Wang, 2020). After vibrio vulnificus is infected, economic animals such as eel (Fouz and Amaro,2003), grouper (Abdullah et al, 2017), sturgeon (Si et al, 2010), tilapia (Chen et al, 2010) and golden pompano (Li et al, 2006) are easy to die in large quantities. Antibiotics are mostly adopted to treat diseases caused by vibrios, and researches show that the vibrios separated from animal diseases detect drug resistance genes and drug resistance of various antibiotics (Kashulin et al, 2017; Letchumanan et al, 2015). With the continuous development of the research and development work of the aquatic vaccine, the application of the vaccine in the diseases of aquaculture is more and more extensive. The traditional inactivated vaccine uses formalin as an inactivating agent, but the complete antigen component is destroyed during inactivation, and the traditional inactivated vaccine has the defects of undesirable immune effect and short immune duration (Sun et al, 2020). The extracellular and surface proteins of Vibrio are readily recognized by infected hosts, have potential as vaccines and can provide immunoprotection (Wang et al, 2017). Meanwhile, research on vibrio vulnificus vaccines in the aspect of aquatic vaccines is relatively less, and development of inactivated vaccines capable of retaining relatively complete antigen structures becomes a development focus of the aquatic inactivated vaccines.

The application No. 201610357120.6, namely Chinese patent application named 'an antibacterial peptide TC38 and application thereof' discloses that the antibacterial peptide TC38 has the effect of killing vibrio vulnificus, but according to the published documents, after C-terminal derived peptides of Tissue Factor Pathway Inhibitors (TFPI) of teleost fish such as sciaenops ocellatus and carps are incubated with the vibrio vulnificus, the rupture of cell structures and the outflow of cell contents are observed by a transmission electron microscope (He et al, 2018; Su et al, 2020), and the rupture of cell structures indicates that the TFPI is difficult to be used for preparing inactivated vaccines. In addition, the current vibrio vulnificus vaccines are mainly combined vaccines (Huang et al, 2012; Shoemaker et al, 2011) of vibrio vulnificus and other pathogenic bacteria, and related researches are relatively few, so that the research and development of an efficient immunological prevention and control method for vibrio pathogens of aquatic animals become an urgent need for the healthy development of the aquaculture industry at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing an creative bacteria innovation inactivated vaccine prepared on the basis of C-terminal derived peptide TC38 derived from TFPI-2 of cynoglossus semilaevis. Based on research finding that after the Vibrio vulnificus is treated by TC38, cell contents of the Vibrio vulnificus are gradually degraded with the passage of time, and cell membranes still keep integrity, the TC38 inactivated vaccine related to the Vibrio vulnificus is prepared, and the relative immune protection rate and immune mechanism induced by the TC38 inactivated vaccine are detected in the turbot body, so that a new way is provided for the research of the Vibrio vulnificus inactivated vaccine.

The invention provides a preparation method of vibrio vulnificus inactivated vaccine, which comprises the following steps:

1) adding TC38 antibacterial peptide into vibrio vulnificus bacterial liquid, and inactivating to obtain inactivated vibrio vulnificus incubation liquid;

2) centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting thallus precipitates to obtain inactivated thallus;

3) and (3) cleaning the inactivated thallus for a plurality of times by using sterile PBS, and dispersing the inactivated thallus in the sterile PBS to obtain the inactivated vaccine.

In one embodiment according to the present invention, the preparation method further comprises:

4) carrying out sterile inspection on the inactivated thallus;

In one embodiment of the invention, the vibrio vulnificus bacterial liquid is prepared by a method comprising the following steps of:

In one embodiment according to the present invention, the TC38 antibacterial peptide is a polypeptide having an amino acid sequence having a homology of not less than 85% with SEQ ID NO. 1.

The invention also provides the vibrio vulnificus inactivated vaccine prepared by the preparation method.

The invention further provides a medicament for preventing or treating the infection of the vibrio vulnificus of the marine cultured animals, which comprises the vibrio vulnificus inactivated vaccine. Preferably, the effective amount is 0.182 mg/strip.

The invention also provides application of the vibrio vulnificus inactivated vaccine in preparation of a medicine for preventing or treating vibrio vulnificus infection of marine cultured animals.

In one embodiment according to the invention the marine farmed animal is selected from one or more of eel, grouper, sturgeon, tilapia, golden pomfret or turbot.

The technical scheme of the invention has the following beneficial effects:

the TC38 antibacterial peptide is used for inactivating vibrio vulnificus to obtain a novel inactivated vaccine, the relative immune protection rate of the vaccine induced in turbots is obviously higher than that of a formalin inactivated vaccine group, the fish body can be induced to generate higher non-specific and specific immune response levels, and the vaccine has a good application prospect in preventing and treating vibrio vulnificus diseases of aquatic animals.

Drawings

FIG. 1 is a statistical representation of cumulative mortality of turbot after challenge;

FIG. 2 is a statistical graph of the changes in the respiratory burst (A) and bactericidal activity (B) of the macrophages of a fry after vaccine immunization;

FIG. 3 is a graph showing the results of the measurement of bactericidal activity of the serum of the vaccine immunized fish;

FIG. 4 is a graph showing the result of measuring the activity of the immune-related enzyme in the turbot serum after vaccination;

FIG. 5 is a graph showing the results of measurement of serum antibody levels and IgM expression levels in head and kidney after vaccination;

FIG. 6 is a graph showing the results of the agglutination assay of Vibrio vulnificus bacteria induced by serum antibodies; wherein, the agglutination phenomenon generated by the serum of TKV group (A), FKV group (B) and PBS group (C) and FITC labeled vibrio vulnificus on the 21 st day of vaccine immunization;

FIG. 7 is a schematic map showing the detection result of the influence of TKV on the expression of the gene related to turbot head kidney tissue immunity.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Test materials, reagents and devices

1. Species and polypeptides:

the Vibrio vulnificus strain is separated from diseased fish, identified and stored in a refrigerator at-80 ℃. TC38(SEQ ID NO:1RNCMEVCVKGEKKHTGQGMIRRLRRNKNNSIFVVRKRV) derived from the C-terminal of the cynoglossus semilaevis TFPI-2 protein. Chemically synthesized by china peptides (shanghai, china). The antimicrobial peptide was purified by high performance liquid chromatography to a purity of > 98%. These lyophilized peptides were stored in an ultra-low temperature refrigerator at-80 ℃ and dissolved in PBS (PH 7.4) at the time of use.

2. Animal breeding:

healthy turbot (average 25 + -1.65 g) was purchased from Harui-sourced aquatic offspring seed cultivation Co., Laoshan mountain area, Qingdao, Ltd.

Before the experiment is carried out, random sampling is needed to detect pathogen-free bacteria before the experiment can be continuously used. During the temporary rearing period, the water temperature of the rearing water is kept at 17 ℃, commercial feed is fed once every noon, and water is changed once.

3. An experimental instrument:

german Kajie tissue Lyser II histiocyte disruption instrument, German BECKMAN high-speed centrifuge, Hangzhou Australian Nano-300 micro spectrophotometer, German Hettich ultra-speed refrigerated centrifuge, American Labnet microporous centrifuge, Shanghai Min spring constant temperature refrigeration shaking table, Beijing Sidoolis electronic balance, Beijing Konghua HH-3A digital display constant temperature water bath, American MD multifunctional enzyme labeling instrument, Beijing six-one DYCP-31BN electrophoresis instrument, Japan TOMYMYSX-500 high pressure sterilization pot, Switzerland LightCycler96 real-time fluorescence quantitative PCR instrument, Qingdao Heil-80 ℃ refrigerator and German Caishi Axio oven 7 full-automatic live cell inverted microscope.

Unless otherwise specified, the experimental values below are expressed as mean ± SE and all statistical analyses are performed using SPSS 20.0 software. Significant differences in experimental values were analyzed using Duncan and LSD variance in one-way analysis of variance (ANOVA) (representing 0.01< p < 0.05;. representing p < 0.01).

EXAMPLE 1 preparation of inactivated vaccine

(1) Recovering Vibrio vulnificus taken out at-80 ℃ to the middle logarithmic growth phase by using a liquid LB culture medium, and then centrifuging, cleaning and resuspending the bacteria in sterile PBS;

(2) inactivating Vibrio vulnificus at 28 deg.C for 6h with 20mg TC38 antibacterial peptide; meanwhile, formalin solution (final concentration of formaldehyde is 3 per mill) is used as a control group;

(3) centrifuging the vibrio vulnificus incubation solutions inactivated in the two modes for 2min at 5000rpm respectively, and cleaning thallus precipitates for 3 times by using sterile PBS respectively;

(4) the inactivated cells were adjusted to 2X 10 with sterile PBS⁸After the concentration of CFU/mL, taking 50 mu L of two groups of inactivated vaccines, coating the two groups of inactivated vaccines in an LB solid culture medium, culturing for 24-48 h at 28 ℃, and carrying out sterile inspection;

(5) if bacteria grow out, TC38 antibacterial peptide is continuously added (formalin solution is continuously added for a control group) for continuous incubation and then cleaning until the bacteria are detected to be qualified, and the prepared inactivated vaccine is temporarily stored at 4 ℃ for later use.

Example 2 Fish immunization method

(1) Randomly dividing 330 turbots into TC38 inactivated groups (TC 38-killd Vibrio vulgaris, TKV), formaldehyde inactivated groups (Formalin-killd Vibrio vulgaris, FKV) and PBS groups, and temporarily culturing the three groups of fries in a culture barrel for more than one week to adapt to the environment;

(2) during the culture period, 10 fish fries are randomly extracted from each group to sample the liver, spleen and kidney of each fish fry, the tissues are ground into homogenate and then coated with a flat plate, and whether the turbot is infected with vibrio vulnificus or not is detected;

(3) injecting 100 μ L of 2 × 10 into abdominal cavity of healthy turbot⁸CFU/mL TKV or FKV vaccine, and 100 μ L sterile PBS injection as control group;

(4) after immunization, fry condition was observed daily and fed with commercial feed, and liver, spleen, kidney and serum were sampled at 1d, 7d, 14d, 21d and 28d after immunization from three groups of turbots and stored at-80 ℃ for subsequent experiments.

Example 3 determination of relative immunoprotection Rate

1. Measurement method

(1) Taking out Vibrio vulnificus from-80 deg.C, recovering to mid-logarithmic growth stage, and resuspending to 5 × 10⁷CFU/mL；

(2) After the three groups of healthy fish fries are stable, 100 mu L of heavy suspension liquid is injected into the abdominal cavity of each fish after one month of vaccine immunization for virus challenge;

(3) recording the death number of three groups of fries every 12h from the challenge day, and analyzing the Cumulative mortality (CSR) and the Relative immune protection Rate (RPS) of the vaccine of each group;

RPS ═ {1- (inactivated group fry mortality%/PBS group fry mortality%) } × 100%.

2. The experimental results are as follows:

to examine the protective effect of the TC38 inactivated vaccine, the Vibrio vulnificus was challenged 4 weeks after the vaccination, and the death was monitored. The results showed that the cumulative mortality rates for the TKV, FKV and PBS fry were 30.77%, 39.53% and 84.62%, respectively (fig. 1), with corresponding RPS of 63.64% and 53.29%, respectively. Compared with the FKV group, the RPS induced by the TKV group is remarkably improved, and the TKV group can generate higher survival rate after vaccination, which shows that the TC38 inactivated vaccine has good protection effect on turbot. Pathogenic separation is carried out on the fish fries after challenge, and the only strain detected in the liver, spleen and kidney of dead fish is vibrio vulnificus, which indicates that the death of the fish is caused by vibrio vulnificus infection.

Example 4 Fish macrophage respiratory burst level and bactericidal Activity

1. Detection of fish macrophage respiratory burst level

(1) Extracting macrophages from fresh spleen and kidney tissues of 5 turbots at 1d, 7d, 14d, 21d and 28d of each group of immunity, and extracting cells according to the flow of a Fish Tissue Macrophage Isolation Kit;

(2) grinding spleen and kidney tissues of each group on a 200-mesh stainless steel screen, and adding a homogenate flushing fluid while grinding;

(3) placing the grinding fluid at room temperature for 2-3h, centrifuging at 2500rpm for 15min, discarding supernatant, and resuspending to 1 × 10 with 1640/double antibody cell culture solution⁷Per mL;

(4) the cell suspension was added in an amount of 100. mu.L to a 96-well cell culture plate, cultured overnight in an incubator at 23 ℃ and washed with sterile PBS to remove nonadherent cells.

(5) Adding 100 mu L of 1mg/mL nitrotetrazolium chloride (NBT), and incubating for 2h at 25 ℃;

(6) removing the culture solution, adding 100% methanol, standing for 10min, removing the supernatant, adding 70% methanol solution, cleaning for 2 times, removing the solution, and naturally air drying 96-well plate at room temperature;

(7) adding 120 mu L of 2M KOH solution into each hole, adding 140 mu L of DMSO to dissolve the blue crystal, and slightly shaking the hole plate to better dissolve the crystal;

(8) the absorbance was measured at 630nm with a microplate reader using KOH/DMSO as a blank control.

2. Macrophage bactericidal index assay

(1) After the macrophages extracted by the method of 3.1.4 are paved on a 96-hole cell culture plate, the culture is carried out for 3 hours, and then culture solution is removed and washed for 2 times by 150 mu L sterile PBS;

(2) culturing Vibrio vulnificus to mid-logarithmic growth phase, centrifuging, washing, and resuspending to 1 × 10 with sterile PBS⁶CFU/mL, adding 100 mu L of resuspension bacterial liquid into each hole, and incubating for 5h at 28 ℃;

(3) 50 μ L of 0.2% Tween20 solution was added to each well to lyse the cells, and 50 μ L of each group was applied to LB solid medium, cultured at 28 ℃ for 12 hours, and colony counting was performed.

3. Serum bactericidal activity assay

(1) Standing turbot venous blood immunized with 1d, 7d, 14d, 21d and 28d at normal temperature for 2h, standing overnight at 4 ℃, centrifuging at 3500rpm/min for 15min, and taking supernatant as serum required by the experiment;

(2) culturing Vibrio vulnificus to mid-logarithmic growth phase and washing with sterile PBS to resuspend to 1 × 10⁷CFU/mL；

(3) Adding 50 mu L of serum of 1d, 7d, 14d, 21d and 28d after each group of immunization into each hole of a 96-hole polypropylene microporous plate, inactivating the serum at 56 ℃ for 30min or not processing, adding 10 mu L of heavy suspension liquid, and incubating for 2h at 28 ℃;

(4) after the above incubation solution was diluted 300 times, 50. mu.L of the diluted solution was applied to a solid LB medium, and cultured at 28 ℃ for 12 to 24 hours, and the colonies were counted.

The survival rate (%) of the bacteria is equal to the colony number of the inactivated group/the colony number of the PBS group multiplied by 100 percent

4. The experimental results are as follows:

head kidney macrophages were collected from fish injected with TC38 inactivated vaccine, formalin inactivated vaccine and PBS after vaccination at 1d, 7d, 14d, 21d and 28d, respectively, for examination of the effect of TC38 inactivated vaccine on macrophage activity. Subsequent respiratory burst activity measurements showed that TKV group fish showed significant respiratory bursts (p <0.05) at 7d, 14d and 21d post inoculation compared to FKV and PBS groups (fig. 2, a). The results of bactericidal activity analysis showed that vibrio vulnificus survival rates at 14d after inoculation were significantly lower in the TKV group of fish than in the FKV and PBS groups (p <0.05) (fig. 2, panel B).

Serum samples were collected at 1d, 7d, 14d, 21d and 28d after inoculation to examine the effect of serum from the TKV group on Vibrio vulnificus bactericidal activity. FIG. 3 shows that the bacterial survival in the TKV group sera was significantly lower than that of the FKV group and PBS group (p <0.05) at 7d, 14d and 28d after inoculation, and that the survival of Vibrio vulnificus in TKV group was 0.494 times that in the FKV group sera at 7 d. Experiments showed that at these time points, the serum bactericidal activity of the TKV group was significantly higher than that of the FKV group (p < 0.05). The survival rate of vibrio vulnificus in the sera of TKV group and FKV group was similar to the control fish sera when the sera were heated before incubation with vibrio vulnificus. Therefore, the novel TC38 inactivated vaccine provided by the invention can better activate macrophages and further activate corresponding immune response reaction, and simultaneously can better promote the secretion of AKP, ACP, T-SOD and lysozyme after being injected with the novel TC38 inactivated vaccine, so as to participate in the nonspecific immune reaction of an organism to achieve the effect of resisting pathogens.

In addition, since the TC38 antibacterial peptide can maintain good inner and outer membrane structures of Vibrio vulnificus, and the vaccine preserves relatively complete bacterial surface antigen structures compared with the formaldehyde inactivated vaccine, the TKV group antibody response speed is high at 7d, 14d, 21d and 28d after inoculation, and the serum antibody level of the Vibrio vulnificus is far higher than that of the FKV group.

EXAMPLE 5 determination of the level of enzyme with immune-related Activity in serum

(1) Alkaline phosphatase (AKP) activity:

AKP activity in serum was measured using Nanjing kit (A059-2). Adding 5 mu L of serum to be detected, phenol standard (0.1mg/mL) and distilled water into a 96-hole enzyme label plate, adding 50 mu L of buffer solution and 50 mu L of matrix solution into each hole, uniformly mixing, carrying out water bath at 37 ℃ for 15min, and then adding 150 mu L of developing solution. Mix gently and measure absorbance at 520nm, and repeat each group five times.

(2) Acid phosphatase (ACP) activity:

measured according to the specification of Nanjing's kit (A060-2), 4. mu.L of serum to be measured, phenol standard (0.1mg/mL) and distilled water are added into a 96-well plate, 40. mu.L of buffer solution and 40. mu.L of matrix solution are added into each well, the mixture is uniformly mixed, water bath is carried out at 37 ℃ for 30min, and then 80. mu.L of alkali liquor and 80. mu.L of developing solution are added. Mixing, standing for 10min, and measuring absorbance at 520 nm.

(3) Total superoxide dismutase (T-SOD) activity:

as xanthine and its oxidase react to produce superoxide anion free radical, it can be oxidized into nitrite which is purple red under the action of colour developing agent. Therefore, the measurement was carried out at 550nm in accordance with the dosages and the order of the reagents indicated in the instructions of Nanjing kit (A001-1).

(4) Lysozyme (Lysozyme) activity:

the lysozyme was measured using Nanjing kit (A050-1-1). Before the experiment, the reagent is bathed to 37 ℃, 0.1mL of serum to be detected and a standard substance (2.5 mu g/mL) are added into a 48-hole enzyme label plate, then 1mL of application bacterial liquid (Micrococcus lysodeikticus) is absorbed and quickly washed into a hole and uniformly mixed, and the absorbance is measured at 530nm at 20s and 2min20s respectively.

The serum used in the above experiments is diluted twice and then measured, and the four enzyme activities are calculated according to the formula indicated in the kit specification.

The results showed that the serum activity of AKP, ACP, lysozyme and T-SOD reached peak in TKV group at 14 days after inoculation, which was significantly higher than that in FKV group and PBS group (p <0.01), which was 1.25, 1.22, 1.43 and 1.23 times that in FKV group, respectively (FIG. 4). In the FKV group, the activity of AKP and ACP was highest at 14D after inoculation (A and B in FIG. 4), but the activity of lysozyme and T-SOD peaked at 21D after inoculation (C and D in FIG. 4). These results indicate that TC38 inactivated vaccines can not only induce higher enzyme activity in serum, but also induce a time advance for lysozyme and T-SOD to peak compared to the FKV group.

Example 6 determination of serum antibody Titers

1. Measurement method

(1) After culturing Vibrio vulnificus to logarithmic growth period, it was washed with sterile PBS and resuspended to 5X 10⁷CFU/mL；

(2) Sera of immunized groups immunized with 7d, 14d, 21d and 28d and control group were mixed with PBS according to 2ⁿCarrying out gradient dilution;

(3) and (3) uniformly mixing 30 mu L of diluted serum and 10 mu L of heavy-suspension bacterium liquid in a 96-well plate, incubating for 3h at 37 ℃, sucking 10 mu L of incubation liquid, dropwise adding the incubation liquid on a glass slide, observing under an upright microscope, and taking visible precipitate generated by the last dilution of the serum as the titer of the agglutination antibody.

2. The experimental results are as follows:

serum antibody levels were measured in the immune group and the control group using an immunoagglutination assay, respectively, and the results showed that antibodies were detected in the sera of both TKV group and FKV group at 7d, 14d, 21d and 28d after vaccination, and that serum antibody levels of TKV group were significantly higher than those of FKV group (p <0.05), 9.97, 4.44, 8.88 and 8.80 times those of FKV group, respectively (a in fig. 5). Subsequent analysis of turbot head kidney IgM expression levels showed that the relative IgM expression levels in the head kidneys of TKV groups were significantly higher than those of FKV groups (p <0.01), 3.71 and 2.11 times higher than those of FKV groups, respectively, at 7d and 21d after vaccination (B in fig. 5).

Example 7 serum agglutination assay

1. Method and procedure

(1) Culturing Vibrio vulnificus to OD₆₀₀Centrifuging, washing and suspending the bacterial liquid in 2mL sterile PBS (0.8), dipping a small amount of Fluorescein Isothiocyanate (FITC) in the suspended bacterial liquid, and incubating for 5h at 28 ℃ in a dark place at 50 rpm;

(2) after 3 centrifugation washes of the incubation, the pellet was resuspended to 1X 10 using sterile PBS⁸CFU/mL, taking 10 mu L serum of each immune 21d and incubating 10 mu L bacterial liquid for 3h at 28 ℃;

(3) 8 mu L of the incubation liquid is fixed on a glass slide, and the agglutination condition is observed by a fluorescence microscope, if the bacterial number under the lens is too much, the agglutination condition can be diluted by a certain multiple for observation.

2. The experimental results are as follows:

the agglutination reaction of Vibrio vulnificus was generated in both TKV group (A in FIG. 6) and FKV group (B in FIG. 6) after 21d of vaccine immunization. The bacterial agglutination level was significantly higher in the TKV group than in the FKV group, and in the control group, there was no bacterial agglutination of Vibrio vulnificus (C in FIG. 6). These results indicate that the serum antibodies were induced by the antigen on the surface of Vibrio vulnificus, and that the binding activity of the serum antibodies of TKV group was higher than that of the serum antibodies of FKV group (FIG. 6).

Analysis of IgM expression level in turbot head kidney shows that relative IgM level in fish head kidney of TKV group is significantly up-regulated compared with FKV group at 7d and 21d after vaccination, and basically corresponds to serum antibody level. In addition, the level and the characteristics of antibodies generated by fish bodies are further verified by incubating FITC labeled vibrio vulnificus and immune 21d serum, and as a result, the TKV group can obviously observe the aggregated vibrio vulnificus, the aggregation effect is higher than that of the FKV group, and the corresponding serum antibodies are induced by the surface antigen of vibrio vulnificus cells. These results indicate that the novel inactivated vaccine of TC38 can not only increase the level of non-specific immune responses, but also enhance specific immune responses such as serum antibody levels.

Example 8 analysis of immune Gene expression

(1) Taking out liver, spleen and kidney tissues stored at-80 ℃, placing on ice, grinding and homogenizing by using tissue grinding fluid, extracting total RNA of the tissues under the instruction of an RNAprep Pure Cell/Bectria Kit, and placing at-80 ℃ for storage;

(2) using a FastKing RT Kit for reverse transcription, taking 8 mu L of total RNA of each group and incubating for 3min at 42 ℃ with 2 mu L of 5 XgDNA Buffer;

(3) mu.L of 10 Xfast RT Buffer, 1. mu.L of RT Enzyme Mix, 2. mu.L of FQ-RT Primer Mix with RNase-Free ddH₂Supplementing O to 10 mu L to prepare Mix;

(4) uniformly mixing the components (2) and (3), incubating at 42 ℃ for 15min, 95 ℃ for 3min and 4 ℃ for 5min, and storing the obtained cDNA at-80 ℃;

(5) SYBR Green Premix Pro Taq HS qPCR Kit is used for amplifying C3, IgM, IL-1 beta, MHC I alpha, MHC II alpha, Mx, NKEF, TCR alpha, TLR3, TNF-alpha, IL-8R, IFN-gamma and CD40, and beta-actin is used as an internal reference gene. The reaction primers are listed in Table 1 and the reaction system is shown in Table 2, using a Roche LightCycler96 system instrument for amplification with a program of 95 ℃ for 30s followed by 40 cycles, 95 ℃ for 5s and 60 ℃ for 30 s.

TABLE 1 primers for quantitative real-time polymerase chain reaction

TABLE 2 fluorescent quantitative PCR reaction System

The influence of TKV immunity on the expression of the genes related to the turbot head and kidney immunity is detected by qRT-PCR, and the result is shown in figure 7, at 21d after immunization, the expression levels of C3, IL-1 beta, MHC II alpha, Mx, NKEF, TCR alpha, TLR3, TNF-alpha and IL-8R in the TKV group in the turbot kidney are obviously higher than those in the FKV group (p is less than 0.01), and are respectively 2.76, 19.29, 2.42, 12.91, 6.11, 2.9, 3.88, 4.30 and 2.33 times of that in the FKV group; there were no significant differences in MHC I α and IFN- γ expression in the three groups (p > 0.05). The above results indicate that TKV induces cytokine production more efficiently than FKV, which may be associated with the ability of TC38 to retain relatively intact antigenic components after treatment of Vibrio vulnificus, thereby enhancing the immunostimulating effect on the body.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Qingdao agricultural university

<120> Vibrio vulnificus inactivated vaccine and preparation method and application thereof

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 38

<212> PRT

<213> Cynoglossus semilaevis (Cynoglossus semilaevis)

<400> 1

Arg Asn Cys Met Glu Val Cys Val Lys Gly Glu Lys Lys His Thr Gly

1 5 10 15

Gln Gly Met Ile Arg Arg Leu Arg Arg Asn Lys Asn Asn Ser Ile Phe

20 25 30

Val Val Arg Lys Arg Val

<210> 2

<211> 20

<212> DNA