CN113694192B - Vibrio vulnificus inactivated vaccine and preparation method and application thereof - Google Patents
Vibrio vulnificus inactivated vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a vibrio vulnificus inactivated vaccine, which comprises the following steps: 1) Adding TC38 antibacterial peptide into the vibrio vulnificus bacterial liquid, and performing inactivation treatment to obtain an inactivated vibrio vulnificus incubation liquid; 2) Centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting bacterial precipitate to obtain an inactivated bacterial; 3) And (3) washing the inactivated thalli with sterile PBS for several times, and dispersing the washed thalli in the sterile PBS to obtain the inactivated vaccine. The invention also provides a corresponding inactivated vaccine and application thereof. The invention uses TC38 antibacterial peptide to inactivate vibrio vulnificus to obtain a novel inactivated vaccine, the relative immune protection rate of the vaccine induced in turbot is obviously higher than that of a formalin inactivated vaccine group, the vaccine can induce fish bodies to generate higher non-specific and specific immune response levels, and the vaccine has good application prospect in preventing and controlling vibrio vulnificus diseases of aquatic animals.
Description
Technical Field
The invention relates to the technical field of animal medicine, in particular to a vibrio vulnificus inactivated vaccine, a preparation method and application thereof.
Background
Vibrio vulnificus is a human and marine zoonotic vibrio, and is mainly of three types: type I organisms, which are prone to ingestion cases and wound infections; type II organisms are pathogenic types causing disease in marine farmed animals; and form iii organisms capable of infecting humans (Wu Houbo and Pan Jinpei, 2001;Bisharat et al, 1999;Kelly and McCormick,1981;Tison et al, 1982). The pathogenicity of vibrio vulnificus is the result of the co-action of a variety of pathogenic factors, including mainly haemolysins, MARTX toxins, siderophores, capsular polysaccharides, lipopolysaccharides, and metalloproteinases etc. (Wang Qingbai, 2018;Li and Wang,2020). After infection with vibrio vulnificus, 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 pomfret (Li et al, 2006) die in large batches. For most diseases caused by vibrio, antibiotics are adopted for treatment, and it has been found that vibrio isolated from a disease animal detects drug resistance genes and drug resistance against various antibiotics (Kashulin et al, 2017;Letchumanan et al, 2015). Along with the continuous development of aquatic vaccine development work, the application of the vaccine in aquaculture diseases is more and more extensive. The conventional inactivated vaccine uses formalin as an inactivating agent, but the whole antigen component is destroyed during inactivation, and has the disadvantages of unsatisfactory immune effect and short immune duration (Sun et al 2020). Extracellular and surface proteins of vibrio are readily recognized by infected hosts, have vaccine potential and can provide immunoprotection (Wang et al, 2017). Meanwhile, the research on vibrio vulnificus vaccines in the aspect of aquatic vaccines is relatively less, and the development of inactivated vaccines capable of retaining relatively complete antigen structures becomes the development focus of aquatic inactivated vaccines.
In chinese patent application No. 201610357120.6, entitled "an antibacterial peptide TC38 and use thereof", it is disclosed that the antibacterial peptide TC38 has an effect of killing vibrio vulnificus, but according to published literature, after incubation of C-terminal derivative peptides of Tissue Factor Pathway Inhibitors (TFPI) of teleomorpha fish, carp and the like with vibrio vulnificus, cell structure disruption, which indicates difficulty in using TFPI for preparation of inactivated vaccine, is observed by transmission electron microscopy (He et al, 2018, su et al, 2020). In addition, current vibrio vulnificus vaccines are mainly of the type of combined vaccines of vibrio vulnificus and other pathogenic bacteria (Huang et al, 2012;Shoemaker et al, 2011) and recombinant vaccines (Kohei et al, 2018), and related researches are relatively few, so that development of an efficient immunological prevention and control method for vibrio vulnificus pathogens of aquatic animals has become an urgent need for healthy development of the current aquaculture industry.
Disclosure of Invention
The invention aims to solve the technical problem of providing an creative bacteria inactivated vaccine prepared based on C-terminal derived peptide TC38 derived from cynoglossus semilaevis TFPI-2. Based on research, after the vibrio vulnificus is treated by TC38, the cell content of the vibrio vulnificus is gradually degraded along with the time, and the cell membrane still keeps integrity, the TC38 inactivated vaccine for the vibrio vulnificus is prepared, the relative immune protection rate and immune mechanism induced by the TC38 inactivated vaccine are detected in turbot bodies, and a new approach is provided for the research of the vibrio vulnificus inactivated vaccine.
The invention provides a preparation method of a vibrio vulnificus inactivated vaccine, which comprises the following steps:
1) Adding TC38 antibacterial peptide into the vibrio vulnificus bacterial liquid, and performing inactivation treatment to obtain an inactivated vibrio vulnificus incubation liquid;
2) Centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting bacterial precipitate to obtain an inactivated bacterial;
3) And (3) washing the inactivated thalli with sterile PBS for several times, and dispersing the washed thalli in the sterile PBS to obtain the inactivated vaccine.
In one embodiment according to the invention, the method of preparation further comprises:
4) Sterile inspection of the inactivated bacteria;
preferably, the sterility test is achieved by a method comprising the steps of: diluting the inactivated thalli with sterile PBS, and then taking a proper amount of the diluted thalli to be coated on a solid culture medium for culturing for 24-48 hours at the temperature of 28 ℃ so as to confirm whether the vibrio vulnificus is completely inactivated.
In one embodiment according to the present invention, the vibrio vulnificus bacterial liquid is prepared by a method comprising the steps of:
resuscitates vibrio vulnificus preserved at-80deg.C into mid-log growth stage with liquid LB culture medium, centrifuging, cleaning, and re-suspending in sterile PBS to obtain the final product; preferably, the concentration of the vibrio vulnificus bacterial liquid obtained after the re-suspension is 2×10 8 CFU/mL。
In one embodiment according to the invention, the TC38 antibacterial peptide is a polypeptide having an amino acid sequence with a homology of not less than 85% to SEQ ID NO. 1.
The invention also provides the vibrio vulnificus inactivated vaccine prepared by the preparation method.
The invention further provides a medicine for preventing or treating vibrio vulnificus infection of marine culture animals, which comprises the inactivated vaccine of vibrio vulnificus. Preferably, the effective amount is 0.182 mg/strip.
The invention also provides application of the vibrio vulnificus inactivated vaccine in preparing a medicament 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 invention uses TC38 antibacterial peptide to inactivate vibrio vulnificus to obtain a novel inactivated vaccine, the relative immune protection rate of the vaccine induced in turbot is obviously higher than that of a formalin inactivated vaccine group, the vaccine can induce fish bodies to generate higher non-specific and specific immune response levels, and the vaccine has good application prospect in preventing and controlling vibrio vulnificus diseases of aquatic animals.
Drawings
FIG. 1 is a statistical diagram of cumulative mortality of turbots after challenge;
FIG. 2 is a statistical graph of changes in macrophage respiratory burst (A) and bactericidal activity (B) of fish fry after vaccine immunization;
FIG. 3 is a graph showing the results of the measurement of the bactericidal activity of the serum of fish fry after immunization with the vaccine;
FIG. 4 is a graph showing the results of the determination of immune-related enzyme activity in turbot serum after vaccination;
FIG. 5 is a graph showing the results of detection of serum antibody levels and IgM expression levels in the head and kidney after vaccination;
FIG. 6 is a graph showing the results of serum antibody-induced bacterial agglutination of Vibrio vulnificus; wherein, on day 21 of vaccine immunization, the agglutination phenomenon is generated by the serum of TKV group (A), FKV group (B), PBS group (C) and FITC marked vibrio vulnificus;
FIG. 7 is a schematic diagram of the detection results showing the effect of TKV on the expression of the turbot head and kidney tissue immune-related genes.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Experimental materials, reagents and apparatus
1. Bacterial species and polypeptides:
vibrio vulnificus strains were isolated from diseased fish and identified and stored in a-80℃refrigerator. TC38 (SEQ ID NO:1 RNCMEVCVKGEKKHTGQGMIRRLRRNKNNSIFVVRKRV) from the C-terminus of the cynoglossus semilaevis TFPI-2 protein. Synthesized chemically by ChinaPeptides company (china, shanghai). The antimicrobial peptides were 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) when used.
2. Breeding animals:
healthy turbots (average 25.+ -. 1.65 g) were purchased from aquatic offspring seed cultivation Co.Ltd in Laoshan mountain area of Qingdao city.
Random sampling is needed to detect pathogen-free bacteria before the experiment is carried out, and the bacteria can be used continuously. During temporary cultivation, the cultivation water temperature was kept at 17 ℃, commercial feed was fed once daily in noon, and water was changed once.
3. Experimental instrument:
the method comprises the steps of a German Kanji tissue Lyseri tissue cell disruptor, a German BECKMAN high-speed centrifuge, a Hangzhou Oriental Nano-300 micro-spectrophotometer, a German Hettich ultracentrifuge, a American Labnet microporous centrifuge, a Shanghai spring constant temperature refrigeration shaker, a Beijing Sidoris electronic balance, a Beijing national HH-3A digital display constant temperature water bath kettle, a American MD multifunctional enzyme-labeling instrument, a Beijing six-DYCP-31 BN electrophoresis instrument, a Japanese TOMYSX-500 autoclave, a Switzerland Roche LightCycler96 real-time fluorescence quantitative PCR instrument, a Qingdao sea-80 ℃ refrigerator and a German Zeiss Axiio observe 7 full-automatic living cell inversion microscope.
Unless otherwise indicated, the following experimental values are expressed as mean ± SE, and all statistical analyses were performed using SPSS 20.0 software. Significant differences in Duncan and LSD analysis of variance experimental values in one-way analysis of variance (ANOVA) were used (representing 0.01< p <0.05; representing p < 0.01).
Example 1 preparation of inactivated vaccine
(1) Resuscitates Vibrio vulnificus taken out at-80 ℃ to mid-log growth period by using a liquid LB culture medium, and then centrifugates and washes bacteria and resuspends the bacteria in sterile PBS;
(2) Inactivating Vibrio vulnificus at 28deg.C for 6 hr using 20mg of TC38 antibacterial peptide; meanwhile, formalin solution (the final concentration of formaldehyde is 3 per mill) is used as a control group;
(3) Centrifuging the inactivated Vibrio vulnificus incubation liquid in the two ways respectively at 5000rpm for 2min, and cleaning the bacterial precipitate with sterile PBS for 3 times;
(4) The inactivated cells were adjusted to 2X 10 with sterile PBS 8 After CFU/mL concentration, two groups of inactivated vaccines of 50 mu L are coated in LB solid medium for culturing for 24-48 h at 28 ℃ and sterile inspection is carried out;
(5) If bacteria grow out, the TC38 antibacterial peptide is continuously added (the formalin solution is continuously added in a control group), the bacteria are continuously incubated and then washed until the bacteria are qualified in sterile test, and the prepared inactivated vaccine is temporarily stored at 4 ℃ for standby.
Example 2 Fish immunization method
(1) Randomly dividing 330 turbots into a TC38 inactivated group (TC 38-killed Vibrio vulnificus, TKV), a formaldehyde inactivated group (formallin-killed Vibrio vulnificus, FKV) and a PBS group, and temporarily culturing three groups of fries in a culture barrel for more than one week to adapt to the environment;
(2) During the cultivation period, 10 fish fries are randomly extracted from each group, the liver, spleen and kidney of the fish fries are sampled, the tissues are ground into homogenate and then are coated on a flat plate, and whether turbot is infected with vibrio vulnificus is detected;
(3) 100 μL 2×10 of healthy turbot is intraperitoneally injected 8 TKV or FKV vaccine of CFU/mL, 100 μl of sterile PBS was injected as control group;
(4) The fish fry were observed daily after immunization and fed commercial feed, and three groups of turbots were sampled for liver, spleen, kidney and serum at 1d, 7d, 14d, 21d and 28d after immunization and stored at-80 ℃ for later use in experiments.
Example 3 determination of relative immune protective Rate
1. Measurement method
(1) Vibrio vulnificus is removed from-80deg.C and resuscitated to mid-log phase, and resuspended to 5×10 7 CFU/mL；
(2) After the three groups of healthy fish fries are stable in state, injecting 100 mu L of resuspension bacteria liquid into each fish abdominal cavity after vaccine immunization for one month to attack toxin;
(3) The number of deaths of three groups of fish fries was recorded every 12h from the day of challenge, and the cumulative mortality of each group (Cumulative mortality rate, CSR) and the relative immunoprotection rate of the vaccine (Relative immune protectionrate, RPS) were analyzed;
RPS = {1- (% mortality of inactivated group fish/% mortality of PBS group fish) } ×100%.
2. Experimental results:
to test the protective effect of TC38 inactivated vaccine, the challenge was performed with vibrio vulnificus 4 weeks after vaccination and its death was monitored. The results showed that the cumulative mortality of fries in TKV, FKV and PBS groups was 30.77%, 39.53% and 84.62%, respectively (fig. 1), and the corresponding RPS was 63.64% and 53.29%, respectively. Compared with FKV group, the RPS induced by TKV group is obviously improved, and TKV group can generate higher survival rate after vaccination, which indicates that TC38 inactivated vaccine has good protective effect on turbot. Pathogen separation is carried out on the fry after the toxicity attack, 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 respiratory burst level and bactericidal Activity of fish macrophages
1. Fish macrophage respiratory burst level detection
(1) Taking 5 fresh spleen and kidney tissues of turbot for macrophage extraction at the 1d, 7d, 14d, 21d and 28d immunization of each group, and extracting cells according to the procedure of Fish Tumor Tissue Macrophage Isolation Kit kit;
(2) Grinding spleen and kidney tissues of each group on a 200-mesh stainless steel screen, and adding a homogenizing flushing liquid while grinding;
(3) Standing the grinding fluid at room temperature for 2-3 hr, centrifuging at 2500rpm for 15min, discarding supernatant, and re-suspending with 1640/double antibody cell culture solution to 1×10 7 individual/mL;
(4) 100. Mu.L of the above cell suspension was added to a 96-well cell culture plate, and the cells were cultured overnight at 23℃in an incubator, and non-adherent cells were removed by washing with sterile PBS.
(5) 100. Mu.L of 1mg/mL nitrotetrazolium chloride (NBT) was added and incubated at 25℃for 2h;
(6) Removing the culture solution, adding 100% methanol, standing for 10min, discarding supernatant, adding 70% methanol solution, cleaning for 2 times, removing the liquid, and naturally air-drying the 96-well plate at room temperature;
(7) 120 mu L of 2M KOH solution is added into each hole, 140 mu L of DMSO is added to dissolve the blue crystal, and the pore plate is slightly vibrated to enable the crystal to be better dissolved;
(8) The absorbance was measured with a microplate reader at a wavelength of 630nm using KOH/DMSO as a blank.
2. Macrophage sterilization index assay
(1) Plating the macrophages extracted according to the method of 3.1.4 on a 96-well cell culture plate, culturing for 3 hours, removing the culture solution, and cleaning with 150 mu L of sterile PBS for 2 times;
(2) Vibrio vulnificus is cultured to mid-logarithmic growth phase, centrifugally washed and resuspended to 1X 10 with sterile PBS 6 CFU/mL, 100 mu L of re-suspension bacteria liquid is added into each hole, and the mixture is incubated for 5 hours at 28 ℃;
(3) Cells were lysed by adding 50. Mu.L of 0.2% Tween20 solution to each well, and each group was incubated in 50. Mu.L of the LB-coated solid medium at 28℃for 12 hours, and colony counts were performed.
3. Serum bactericidal activity assay
(1) Standing turbot venous blood immunized for 1d, 7d, 14d, 21d and 28d at normal temperature for 2h, standing overnight at 4 ℃, centrifuging at 3500rpm/min for 15min, and taking the supernatant as serum required by experiments;
(2) Vibrio vulnificus is cultured to mid-log phase and resuspended to 1×10 with sterile PBS wash 7 CFU/mL；
(3) Adding 50 mu L of serum of 1d, 7d, 14d, 21d and 28d after immunization into each well of a 96-well polypropylene micro-well plate, inactivating the serum at 56 ℃ for 30min or not, adding 10 mu L of re-suspension bacteria liquid, and incubating at 28 ℃ for 2h;
(4) After 300 times dilution of the incubation liquid, 50 mu L of the dilution liquid is coated in a solid LB culture medium, and the culture is carried out at 28 ℃ for 12-24 hours, so that colony counting is carried out.
Bacterial survival (%) = number of inactivated group colonies/PBS group colonies x 100%
4. Experimental results:
the cephalopod 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 breath burst activity assays showed that fish in group TKV showed significant breath bursts (p < 0.05) at 7d, 14d and 21d post-inoculation compared to fish in FKV and PBS groups (a in figure 2). The results of the bactericidal activity analysis showed that the vibrio vulnificus survival rate at 14d post-inoculation was significantly lower for the TKV group of fish than for the FKV and PBS groups of fish (p < 0.05) (B in fig. 2).
Serum samples were collected at 1d, 7d, 14d, 21d and 28d post inoculation to examine the effect of group TKV serum on vibrio vulnificus bactericidal activity. FIG. 3 shows that bacterial viability in the sera of TKV groups 7d, 14d and 28d after inoculation was significantly lower than that of FKV and PBS groups (p < 0.05), and that the viability of Vibrio vulnificus in the 7d TKV group was 0.494-fold that in the sera of FKV group. Experiments showed that at these time points, the serum bactericidal activity of group TKV was significantly higher than that of FKV group (p < 0.05). The viability of Vibrio vulnificus in the sera of TKV and FKV groups when heated prior to incubation with Vibrio vulnificus was similar to that of control fish. Therefore, the TC38 novel inactivated vaccine provided by the invention can better activate macrophages to activate corresponding immune response, and meanwhile, after the TC38 novel inactivated vaccine is injected, the secretion of AKP, ACP, T-SOD and lysozyme can be better promoted, and the TC38 novel inactivated vaccine is further participated in the nonspecific immune response of an organism, so that the effect of resisting pathogens is achieved.
In addition, since the vaccine prepared from the TC38 antibacterial peptide can maintain a good inner and outer membrane structure of Vibrio vulnificus, it maintains a relatively complete bacterial surface antigen structure compared with the formaldehyde inactivated vaccine, thus the TKV group antibodies respond very fast after inoculation at 7d, 14d, 21d and 28d, and the serum antibody level against Vibrio vulnificus is far higher than that of FKV group.
EXAMPLE 5 determination of the level of immune-related active enzyme in serum
(1) Alkaline phosphatase (Alkaline phosphatase, AKP) activity:
AKP activity in serum was measured using the nanjing built kit (a 059-2). 5 mu L of serum to be detected, phenol standard (0.1 mg/mL) and distilled water are added into a 96-well ELISA plate, 50 mu L of buffer solution and 50 mu L of matrix solution are added into each well, the mixture is uniformly mixed, water bath is carried out for 15min at 37 ℃, and then 150 mu L of chromogenic solution is added. The absorbance was measured at 520nm with gentle mixing and repeated five times for each group.
(2) Acid phosphatase (ACP) activity:
according to the specification of Nanjing's kit (A060-2), adding 4. Mu.L of serum to be tested, phenol standard (0.1 mg/mL) and distilled water into 96-well plate, adding 40. Mu.L of buffer solution and 40. Mu.L of matrix solution into each well, mixing uniformly, then adding 80. Mu.L of alkali solution and 80. Mu.L of color development solution into the mixture in water bath at 37 ℃ for 30 min. The mixture was gently mixed, and after resting for 10min, the absorbance was measured at 520 nm.
(3) Total superoxide dismutase (Total superoxide dismutase, T-SOD) activity:
because xanthine and its oxidase react to produce superoxide anion radical, it can oxidize to nitrite which appears purple under the condition of color-developing agent. Thus, the doses and order of the reagents indicated in the specification of the Nanjing's kit (A001-1) were measured at 550 nm.
(4) Lysozyme (Lysozyme) activity:
lysozyme was measured using the Nanjing build kit (A050-1-1). Before the experiment, the reagent is put in a water bath to 37 ℃, 0.1mL of serum to be detected and a standard substance (2.5 mug/mL) are added into a 48-hole ELISA plate, then 1mL of application bacterial liquid (Micrococcus lysodeikticus) is absorbed and rapidly poured into the hole and evenly mixed, and the absorbance value is measured at 530nm at 20s and 2min for 20s respectively.
The serum used in the above experiment was diluted twice and measured, and four enzyme activities were calculated according to the formula indicated in the kit instructions.
The results showed that AKP, ACP, lysozyme and T-SOD activity peaked in the TKV group serum 14 days post-inoculation, significantly higher than the FKV and PBS group plots (p < 0.01), 1.25, 1.22, 1.43 and 1.23 fold, respectively, of the FKV group (fig. 4). In the FKV group, the activities of AKP and ACP were highest at 14D after inoculation (A and B in FIG. 4), but the activities of lysozyme and T-SOD peaked at 21D after inoculation (C and D in FIG. 4). These results indicate that TC38 inactivated vaccines can induce not only higher enzymatic activity in serum, but also a time advance peak of lysozyme and T-SOD compared to FKV groups.
EXAMPLE 6 determination of serum antibody titers
1. Measurement method
(1) After Vibrio vulnificus is cultured to mid-log phase, the culture is washed with sterile PBS and resuspended to 5×10 7 CFU/mL；
(2) Immunization groups 7d, 14d, 21d and 28d will be immunizedPBS was used for control group serum at 2 n Carrying out gradient dilution;
(3) Mixing 30 μl of diluted serum with 10 μl of resuspended bacteria solution in 96-well plate, incubating at 37deg.C for 3 hr, absorbing 10 μl of the incubation solution, dripping onto glass slide, and observing under a normal microscope to obtain visible precipitate as agglutination antibody titer.
2. Experimental results:
serum antibody levels of the immunized and control groups were tested by immunoagglutination experiments, respectively, and the results showed that antibodies were detected in the serum of both TKV and FKV groups at 7d, 14d, 21d and 28d, and that the serum antibody level of TKV was significantly higher than that of FKV group (p < 0.05), 9.97, 4.44, 8.88 and 8.80 times that of FKV group (a in fig. 5), respectively. Subsequent analysis of turbot head kidney IgM expression levels showed that the relative IgM expression levels in the fish head kidney of tkv groups were significantly higher than in FKV groups (p < 0.01) at both 7d and 21d after vaccination, 3.71 and 2.11 fold higher than in FKV groups (B in fig. 5), respectively.
EXAMPLE 7 serum agglutination assay
1. Method and procedure
(1) Culturing Vibrio vulnificus to OD 600 =0.8, the bacterial solution was centrifuged, washed and resuspended in 2mL sterile PBS, a small amount of Fluorescein Isothiocyanate (FITC) was dipped in the resuspended bacterial solution and incubated at 28 ℃ in the dark at 50rpm for 5h;
(2) After centrifugation of the incubation for 3 times, the pellet was resuspended to 1X 10 using sterile PBS 8 CFU/mL, taking 10 mu L serum of each group of immunization 21d and incubating with 10 mu L bacterial liquid at 28 ℃ for 3h;
(3) 8 mu L of incubation liquid is fixed on a glass slide, and the aggregation condition is observed by a fluorescence microscope, and if the bacterial quantity under the lens is excessive, the bacteria can be diluted by a certain multiple for observation.
2. Experimental results:
the vaccine was 21d later in both TKV (A in FIG. 6) and FKV (B in FIG. 6) groups, allowing the Vibrio vulnificus to generate an agglutination reaction. The TKV group had significantly higher levels of bacterial aggregation than the FKV group, and in the control group, there was no bacterial aggregation 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 the binding activity of the serum antibodies of group TKV was higher than that of the serum antibodies of FKV group (fig. 6).
Analysis of IgM expression levels in turbot head kidneys showed that relative IgM levels in head kidneys of group TKV fish bodies were significantly up-regulated compared to FKV groups at 7d and 21d post-vaccination, essentially corresponding to serum antibody levels. In addition, the levels and characteristics of antibodies produced by fish bodies were further verified by incubation of FITC-labeled Vibrio vulnificus with serum of immunization 21d, and as a result, the TKV group can obviously observe agglomerated Vibrio vulnificus, and the agglutination effect is higher than that of the FKV group, which indicates that the corresponding serum antibodies are induced by surface antigens of Vibrio vulnificus cells. These results indicate that the novel TC38 inactivated vaccine can not only increase the level of non-specific immune responses, but also enhance specific immune responses such as serum antibody levels.
Example 8 immune Gene expression analysis
(1) Taking out liver, spleen and kidney tissue preserved at-80deg.C, placing on ice, homogenizing with tissue grinding liquid, extracting total RNA of tissue under the direction of RNAprep Pure Cell/Becteria Kit, and placing at-80deg.C;
(2) Reverse transcription Using FastKing RT Kit, groups of 8. Mu.L total RNA were incubated with 2. Mu.L 5 XgDNA Buffer at 42℃for 3min;
(3) According to 2. Mu.L of 10 XFast RT Buffer, 1. Mu.L of RT Enzyme Mix, 2. Mu.L of FQ-RT Primer Mix and using Rnase-Free ddH 2 O is added to 10 mu L to prepare Mix;
(4) Uniformly mixing the cDNA obtained in the step (2) and the cDNA obtained in the step (3), incubating at 42 ℃ for 15min,95 ℃ for 3min and 4 ℃ for 5min, and preserving the cDNA at-80 ℃;
(5) The SYBR Green Premix Pro Taq HS qPCR Kit kit was used for amplification of 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 the reference gene was beta-actin. The reaction primers are listed in Table 1 and the reaction system is shown in Table 2, and amplification is performed using a Roche light cycler96 system instrument, with a procedure of 95℃for 30s followed by 40 cycles, 95℃for 5s and 60℃for 30s.
TABLE 1 primers for quantitative real-time polymerase chain reaction
TABLE 2 fluorescent quantitative PCR reaction System
The effect of the qRT-PCR assay TKV immunization on the expression of the turbot head and kidney immune related genes is shown in FIG. 7, and the results show that the expression level of C3, IL-1 beta, MHC II alpha, mx, NKEF, TCR alpha, TLR3, TNF-alpha and IL-8R in TKV groups of turbot kidney is significantly higher than that of FKV group (p < 0.01), 2.76, 19.29, 2.42, 12.91, 6.11, 2.9, 3.88, 4.30 and 2.33 times that of FKV group respectively, 21d after immunization; there was no significant difference in the expression of three groups of MHC iα and IFN- γ (p > 0.05). The above results indicate that TKV is more effective in inducing cytokine production relative to FKV, which may be associated with the ability of TC38 to retain relatively intact antigenic components after treatment of vibrio vulnificus, thereby enhancing the immune stimulating effect on the body.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (5)
1. A method for preparing a vibrio vulnificus inactivated vaccine, comprising the steps of:
1) Adding TC38 antibacterial peptide into the vibrio vulnificus bacterial liquid, and performing inactivation treatment to obtain an inactivated vibrio vulnificus incubation liquid;
2) Centrifuging the inactivated vibrio vulnificus incubation liquid, and collecting bacterial precipitate to obtain an inactivated bacterial;
3) Washing the inactivated thalli with sterile PBS for several times, and dispersing the washed thalli in the sterile PBS to obtain an inactivated vaccine;
the amino acid sequence of the TC38 antibacterial peptide is shown as SEQ ID NO. 1.
2. The method of manufacturing as set forth in claim 1, further comprising:
sterile inspection of the inactivated bacteria;
the sterility test is achieved by a method comprising the steps of: and diluting the inactivated thalli with sterile PBS, and then taking a proper amount of the diluted thalli to be coated on a solid culture medium for culturing for 24-48 hours at the temperature of 28 ℃ so as to confirm whether the vibrio vulnificus is completely inactivated.
3. The preparation method of claim 1, wherein the vibrio vulnificus bacterial liquid is prepared by a method comprising the following steps:
resuscitates vibrio vulnificus preserved at-80deg.C into mid-log growth stage with liquid LB culture medium, centrifuging, cleaning, and re-suspending in sterile PBS to obtain the final product; the concentration of the vibrio vulnificus bacterial liquid obtained after the resuspension is 2 multiplied by 10 8 CFU/mL。
4. The inactivated vaccine for Vibrio vulnificus prepared by the preparation method according to any one of claims 1 to 3.
5. The use of the inactivated vaccine for Vibrio vulnificus according to claim 4 for the preparation of a medicament for preventing infection by Vibrio vulnificus in turbot.
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| Han-Ning Huang等.Modulation of immune responses by the antimicrobial peptide, epinecidin (Epi)-1, and establishment of an Epi-1-based inactivated vaccine.Biomaterials.2011,第32卷(第14期),第3627-3636页. * |
| Min Zhang等.TC38, a teleost TFPI-2 peptide that kills bacteria via penetration of the cell membrane and interaction with nucleic acids.Fish Shellfish Immunol.2017,摘要和第105页. * |
| Qin-Qin Gu等.Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.Fish Shellfish Immunol.2021,第197-204页. * |
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