CN117298262A - Inactivated fragrant fish pseudomonas vaccine and preparation and application thereof - Google Patents
Inactivated fragrant fish pseudomonas vaccine and preparation and application thereof Download PDFInfo
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- CN117298262A CN117298262A CN202310766194.5A CN202310766194A CN117298262A CN 117298262 A CN117298262 A CN 117298262A CN 202310766194 A CN202310766194 A CN 202310766194A CN 117298262 A CN117298262 A CN 117298262A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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Abstract
The invention relates to the technical field of immunology, in particular to an inactivated and fragrant fish pseudomonas vaccine and preparation and application thereof. The inactivated pseudomonas vaccine for killing the fragrant fish comprises the concentration of 5 multiplied by 10 6 CFU/mL～3×10 8 The method comprises the steps of (1) inactivating liquid of pseudomonas for killing the fragrant fishes in CFU/mL and chitosan oligosaccharide with the concentration of 0.05 mg/mL-50 mg/mL; the invention discloses a vaccine containing inactivated pseudomonas fragi and chitosan oligosaccharide for the first time. Vaccine prepared by the pseudomonas inactivated liquid for killing the fish and chitosan oligosaccharide according to reasonable proportion is proved to have good immune effect through experiments aiming at various fishesThe method has the advantages of lasting and effective immune protection effect, no residue in fish body, easy administration, low preparation cost, and simple and practical immune method.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to an inactivated and fragrant fish pseudomonas vaccine and preparation and application thereof.
Background
In recent years, with the continuous expansion of aquaculture cultivation scale, high-density cultivation is common, and large-scale outbreaks of cultivation diseases are unavoidable. The loss of fish infectious diseases caused by each year accounts for over 20 percent of the yield, and the diseases with large-scale outbreaks become the main obstacle in the aquaculture industry.
The main causes of infectious diseases in fish include bacteria, parasites and viruses. Large yellow croaker (Larimichthys crocea) is an important economic fish in China, and the healthy development of the cultivation industry is severely restricted by cultivation diseases along with the increase of cultivation density and the expansion of scale. Visceral ichthyophthiriasis (Visceral granulomas disease, VGD) is a major bacterial disease in the cultivation process of large yellow croaker, has high mortality and incidence rate, and the pathogenic bacteria of the visceral ichthyophthiriasis are pseudomonas fragicide (Pseudomonas plecoglossicida). Currently, vaccination is an effective way to prevent bacterial diseases, and thus, wu needs to develop vaccine candidate products against visceral ichthyophthiriasis of large yellow croakers. The inactivated vaccine has high safety and short research and development period, and is the first choice for developing the vaccine for fish. Visceral ichthyophthiriasis has the characteristics of wide popularity, high morbidity, high mortality and the like, and brings great threat to marine cultured fishes.
At present, chemical synthesized medicines or antibiotics are generally used for controlling the large-scale outbreak of the disease in mariculture production, but long-term administration has a plurality of defects: 1) Sometimes the etiology of fish diseases is complex, a large amount of medicines are blindly used, and a plurality of diseases can not be treated by targeted medicines at present; 2) The chemical medicine has weak prevention effect, and ill fish can not ingest effective medicine dosage because of poor ingestion; 3) Drugs ingested by fish often remain therein, endangering human health, and long-term administration causes environmental pollution and causes the appearance of drug resistance of a large number of microorganisms.
In addition, under the maintenance of long-term medicaments, a large number of obsolete individuals with weak disease resistance survive to influence the quality of fish meat, and the disease resistance of the whole population is reduced to seriously influence the development of the mariculture industry.
Therefore, the development of fish vaccines for preventing bacterial diseases by using immunological techniques has become an important concern for researchers worldwide. At present, the prevention and treatment of visceral ichthyophthiriasis mainly depend on a pseudomonas vaccine for killing the fragrant fish, wherein the pseudomonas vaccine for killing the fragrant fish, the attenuated live vaccine for killing the pseudomonas for killing the fragrant fish and the multivalent vaccine for resisting the pseudomonas for killing the fragrant fish are studied more, and the immune effect is uneven. In addition, there are certain toxic and side effects, for example, some vaccine adjuvants are found to remain in fish bodies (such as viscera), which threatens the safety of people eating.
In summary, there is an urgent need in the art to develop novel well-developed immune vaccines that provide farmed fish with good immunity against pseudomonas fragicide.
Disclosure of Invention
In order to solve the problems, the invention aims to provide an inactivated and fragrant fish pseudomonas vaccine and a preparation method and application thereof.
The aim of the invention can be achieved by the following technical scheme:
a first object of the present invention is to provide an inactivated pseudomonas for aromaticalis, which comprises an inactivated pseudomonas for aromaticalis liquid and chitosan oligosaccharide;
in the vaccine, the concentration of the pseudomonas killing liquid for killing the fragrant fish is 5 multiplied by 10 6 CFU/mL～3×10 8 CFU/mL, the concentration of chitosan oligosaccharide is 0.05 mg/mL-50 mg/mL.
In one embodiment of the invention, the concentration of the pseudomonas fragi-killing inactivated solution in the vaccine is 1×10 7 CFU/mL～1.5×10 8 CFU/mL, the concentration of chitosan oligosaccharide is 0.1 mg/mL-10 mg/mL.
In one embodiment of the invention, the concentration of the pseudomonas fragi-killing inactivated solution in the vaccine is 2×10 7 CFU/mL～8×10 7 CFU/mL, the final concentration of chitosan oligosaccharide is 0.5 mg/mL-5 mg/mL.
In one embodiment of the invention, the chitosan oligosaccharide has an average molecular weight of 2000 and a degree of deacetylation higher than 90%;
the preservation number of the pseudomonas for killing the fragrant fish is CGMCC No.8985.
The second object of the invention is to provide a method for preparing an inactivated pseudomonas for killing fish, comprising the following steps:
(S1) inoculating activated pseudomonas for killing the fragrant fish into a liquid culture medium for pre-culturing, and then adding formaldehyde for inactivating treatment to obtain pseudomonas for killing the fragrant fish inactivating liquid;
(S2) after the post-treatment of the pseudomonas putida inactivating liquid prepared in the step (S1) is uniformly mixed with the chitosan oligosaccharide solution, so as to obtain the pseudomonas putida inactivating vaccine.
In one embodiment of the present invention, in step (S1), the liquid medium is TSB liquid medium, and the concentration of formaldehyde is lower than 0.5% by volume.
In one embodiment of the present invention, in the step (S1), the temperature during the preculture is 30 ℃ for 14 hours;
the post-treatment is dilution with physiological seawater;
in the inactivation treatment process, the temperature is 28 ℃, the time is 48 hours, and the bacterial liquid is fully stirred for 1 time every 6 hours.
In one embodiment of the invention, the pseudomonas fragi is inoculated in an amount of 1% by volume.
In one embodiment of the invention, the plate is used for culturing the pseudomonas pseudosciaena, light yellow clones are picked up in a TSB liquid culture medium, and the pseudomonas pseudosciaena is cultured for 12 hours at 30 ℃ to complete the activation of the pseudomonas pseudosciaena.
In one embodiment of the invention, the physiological seawater comprises NaCl, mgCl 2 ·6H 2 O、MgSO 4 ·7H 2 O、KCl、NaHCO 3 、CaCl 2 ·2H 2 O；
Wherein the concentration of NaCl is 20g/L, mgCl 2 ·6H 2 The concentration of O is 4.8g/L, mgSO 4 ·7H 2 The concentration of O is 3.5g/L, the concentration of KCl is 0.7g/L, and the concentration of NaHCO is 0.7g/L 3 At a concentration of 0.11g/L CaCl 2 ·2H 2 The concentration of O was 1.21g/L.
The third object of the invention is to provide an application of the inactivated and fragrant fish pseudomonas vaccine in preparing a composition for preventing pathogenic bacteria infection of fishes.
A fourth object of the present invention is to provide a composition for preventing pathogenic bacterial infection in fish, comprising the inactivated pseudomonas fish vaccine described above;
the pathogenic bacteria are selected from one or more of Pseudomonas fragi, aeromonas salmonicida or Aeromonas sulunder.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the chitosan oligosaccharide and the pseudomonas killing inactivating liquid are mixed in a proper proportion, so that the immunity protection can be obviously improved, high-titer antibodies can be obtained in the fish body, and a lasting and effective immunity protection effect can be achieved.
(2) The chitosan oligosaccharide can be dissolved in an aqueous solvent, is convenient to administer and is beneficial to large-scale application.
(3) The chitosan oligosaccharide is easy to be absorbed and metabolized by fish, and has no residue in fish body.
(4) The inactivated pseudomonas fish vaccine provided by the invention has the advantages of low preparation cost, simple immunization method and easiness in actual operation.
Drawings
FIG. 1 is an analysis of the immunopotentiating effect of chitosan oligosaccharide alone.
FIG. 2 shows the relative immunoprotection of P.species of wild aromaticillium species by injection of P.species of aromaticillium species inactivation solution and chitosan oligosaccharide into each immune group for 1 month.
FIG. 3 shows the relative immunoprotection of P.aromaticum from wild P.aromaticum by injecting immune groups of chitosan oligosaccharides to immune yellow croakers for 4, 8 and 12 weeks.
FIG. 4 shows serum antibody levels and cross-immune analysis of P.aromaticillium killed and 4 weeks of immune groups injected with chitosan oligosaccharide.
FIG. 5 shows serum antibody levels and cross-immune analysis of 8 weeks of immune groups of P.aromaticilli inactivated solution and chitosan oligosaccharide injected immune to large yellow croaker.
FIG. 6 shows serum antibody levels and cross-immune analysis of immune groups of P.aromaticis inactivated liquid and chitosan oligosaccharide injected with immune groups of yellow croaker at 12 weeks.
FIG. 7 shows adjuvant residues in the immunized and control groups after 3 months of immunization with the Pseudomonas killing liquid and adjuvant.
Detailed Description
The invention provides an inactivated pseudomonas vaccine for killing fish, which comprises an inactivated pseudomonas liquid for killing the fish and chitosan oligosaccharide;
in the vaccine, the concentration of the pseudomonas killing liquid for killing the fragrant fish is 5 multiplied by 10 6 CFU/mL～3×10 8 CFU/mL, the concentration of chitosan oligosaccharide is 0.05 mg/mL-50 mg/mL.
In one embodiment of the invention, the concentration of the pseudomonas fragi-killing inactivated solution in the vaccine is 1×10 7 CFU/mL～1.5×10 8 CFU/mL, the concentration of chitosan oligosaccharide is 0.1 mg/mL-10 mg/mL.
In one embodiment of the invention, the concentration of the pseudomonas fragi-killing inactivated solution in the vaccine is 2×10 7 CFU/mL～8×10 7 CFU/mL, the final concentration of chitosan oligosaccharide is 0.5 mg/mL-5 mg/mL.
In one embodiment of the invention, the chitosan oligosaccharide has an average molecular weight of 2000 and a degree of deacetylation higher than 90%;
the preservation number of the pseudomonas for killing the fragrant fish is CGMCC No.8985.
The invention provides a preparation method of an inactivated fragrant fish pseudomonas vaccine, which comprises the following steps:
(S1) inoculating activated pseudomonas for killing the fragrant fish into a liquid culture medium for pre-culturing, and then adding formaldehyde for inactivating treatment to obtain pseudomonas for killing the fragrant fish inactivating liquid;
(S2) after the post-treatment of the pseudomonas putida inactivating liquid prepared in the step (S1) is uniformly mixed with the chitosan oligosaccharide solution, so as to obtain the pseudomonas putida inactivating vaccine.
In one embodiment of the present invention, in step (S1), the liquid medium is TSB liquid medium, and the concentration of formaldehyde is lower than 0.5% by volume.
In one embodiment of the present invention, in the step (S1), the temperature during the preculture is 30 ℃ for 14 hours;
the post-treatment is dilution with physiological seawater;
in the inactivation treatment process, the temperature is 28 ℃, the time is 48 hours, and the bacterial liquid is fully stirred for 1 time every 6 hours.
In one embodiment of the invention, the pseudomonas fragi is inoculated in an amount of 1% by volume.
In one embodiment of the invention, the plate is used for culturing the pseudomonas pseudosciaena, light yellow clones are picked up in a TSB liquid culture medium, and the pseudomonas pseudosciaena is cultured for 12 hours at 30 ℃ to complete the activation of the pseudomonas pseudosciaena.
In one embodiment of the invention, the physiological seawater comprises NaCl, mgCl 2 ·6H 2 O、MgSO 4 ·7H 2 O、KCl、NaHCO 3 、CaCl 2 ·2H 2 O；
Wherein the concentration of NaCl is 20g/L, mgCl 2 ·6H 2 The concentration of O is 4.8g/L, mgSO 4 ·7H 2 The concentration of O is 3.5g/L, the concentration of KCl is 0.7g/L, and the concentration of NaHCO is 0.7g/L 3 At a concentration of 0.11g/L CaCl 2 ·2H 2 The concentration of O was 1.21g/L.
The invention provides application of the inactivated and fragrant fish pseudomonas vaccine in preparing a composition for preventing pathogenic bacteria infection of fish.
The invention provides a composition for preventing fish pathogenic bacteria infection, which comprises the inactivated and fragrant fish pseudomonas vaccine;
the pathogenic bacteria are selected from one or more of Pseudomonas fragi, aeromonas salmonicida or Aeromonas sulunder.
The invention discloses a vaccine containing inactivated pseudomonas fragi and chitosan oligosaccharide for the first time. Tests on various fishes prove that the vaccine prepared by the pseudomonas inactivated liquid for killing the fragrant fishes and the chitosan oligosaccharide according to reasonable proportions has good immune effect, can achieve lasting and effective immune protection effect, has no residue in the fish body, is easy to administer, has low preparation cost, and is simple and easy to operate in practice.
The choice of immunoadjuvant is a very important factor in the preparation of vaccines. Some immunoadjuvants, although having an effect of promoting the immunogenicity of an immunogen, have some toxic side effects such as remaining in fish or viscera. Some adjuvants are difficult to inject and administer because of the dosage form relationship (such as oily adjuvants), and are not suitable for administration of large-scale farmed fish. The invention provides a novel vaccine comprising inactivated pseudomonas putida and chitosan oligosaccharide, wherein the pseudomonas putida and the chitosan oligosaccharide are mixed in a reasonable proportion. The vaccine has less residue in vivo, easy administration and good immune effect.
The chitosan oligosaccharide is also called chitosan oligosaccharide and oligomeric chitosan, and is an oligosaccharide product with the polymerization degree of 2-20, which is obtained by degrading chitosan through a degradation technology (such as a biological enzyme technology or a microwave degradation technology). The water solubility is better.
In the prior art, chitosan oligosaccharide is generally applied to the fields of food additives, feed additives, daily chemical industry and the like. In the pharmaceutical or veterinary field, it has not been used as a precedent for immunoadjuvant.
In a preferred embodiment of the present invention, the chitosan oligosaccharide has an average molecular weight of about 2000, a degree of deacetylation of more than 90%, and is readily soluble in water. During preparation, the solution of chitosan oligosaccharide prepared by using physiological seawater can be filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m for later use.
As a preferred mode of the invention, the vaccine prepared by mixing the chitosan oligosaccharide and the inactivated pseudomonas fish, has particularly ideal immune effect and good promoting effect on humoral immunity.
Pseudomonas fragi is a species known in the art to cause infectious diseases in fish. In the invention, the inactivated pseudomonas for killing the fragrant fish also comprises the attenuated pseudomonas for killing the fragrant fish.
Methods known in the art can be used to prepare inactivated pseudomonas fragi or attenuated pseudomonas fragi. As a preferred mode of the invention, the pseudomonad of the aromaticacid is inactivated with formaldehyde (preferably the final formaldehyde concentration is not more than 0.5%), diluted with physiological seawater and then mixed with the chitosan oligosaccharide solution. More specifically, the method for inactivating pseudomonas for aromaticalis comprises the following steps: culturing Pseudomonas fragi by plate, picking pale yellow monoclonal in TSB liquid culture medium, and culturing at 30deg.C for 12 hr; then inoculating the strain into TSB liquid culture medium according to the inoculation amount of 1% by volume, culturing for 14h at 30 ℃, adding formaldehyde with the final concentration of 0.5%, inactivating for 48h at 28 ℃, and fully stirring 1 bacterial liquid every 6h during the period.
The invention also provides a composition, preferably a vaccine, wherein the vaccine is a vaccine which is obtained by mixing the pseudomonas killing liquid and a pharmaceutically acceptable carrier and can be used for preventing pathogenic bacteria infection. The pharmaceutically acceptable carrier is chitosan oligosaccharide. The vaccine can be used for fish, and can be generally prepared into injection.
In the present invention, the term "pathogenic bacteria" refers to a type of bacteria that infects fish. As a preferred mode of the invention, the pathogenic bacteria include, but are not limited to: pseudomonas fragi (Pseudomonas plecoglossicida), aeromonas salmonicida (Aeromonas salmonicide), aeromonas sulburet (Aeromonas schubertii).
As a preferred mode of the present invention, the vaccine may comprise: the pseudomonas inactivated solution for killing the fragrant fish and an effective amount of chitosan oligosaccharide are used as an immunoadjuvant.
As a preferred mode of the invention, the final concentration of the inactivated Pseudomonas fragi is 5×10 in the vaccine 6 CFU/mL～3×10 8 CFU/mL; preferably 1X 10 7 CFU/mL～1.5×10 8 CFU/mL; more preferably 2X 10 7 CFU/mL～8×10 7 CFU/mL; the final concentration of the chitosan oligosaccharide is 0.02 mg/mL-100 mg/mL; preferably 0.05mg/mL to 50mg/mL; more preferably 0.1mg/mL to 10mg/mL (still more preferably 0.5mg/mL to 5 mg/mL). The compound has good administration effect under the concentration, and can generate antibodies with very high titers in fish bodies.
The method for preparing the pseudomonas for killing the salmon has remarkable immune effect, is proved on zebra fish and large yellow croaker, obviously improves serum antibody titer of the large yellow croaker after immunization, and has a certain cross immune protection effect on the pseudomonas for killing the salmon and the pseudomonas for killing the Shubert.
In a specific embodiment of the invention, sterilized physiological seawater is used for dilution to the required concentration of the injection zebra fish and the large yellow croaker for later use. On zebra fish, the final concentration of the pseudomonas inactivated solution for killing the fragrant fish is controlled to be 8 multiplied by 10 according to the method 7 CFU/mL,2mg/mL chitosan oligosaccharide are mixed in equal volume, zebra fish is injected at 5 mu L/tail, immunization is carried out for one month, and the immune protection effect can reach about 90%; on large yellow croaker, the final concentration of the Pseudomonas killing liquid is controlled to be 4×10 according to the method 8 CFU/mL, chitosan oligosaccharide is prepared into 2mg/mL by using physiological seawater, the chitosan oligosaccharide and the physiological seawater are mixed in equal volume, and the immune protection effect of three months can reach 100% after 0.1 mL/tail injection of large yellow croaker. The addition of chitosan oligosaccharide obviously improves the serum antibody level and enhances the humoral immunity effect of the host.
The vaccine of the present invention may be prepared in the form of a kit or kit for use by those skilled in the art. Optionally including instructions for use therein.
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, unless otherwise specified, all reagents used were commercially available, and all detection means and methods used were conventional in the art.
In the examples described below, the experimental methods without specifying the specific conditions are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Example 1
The embodiment provides an inactivated and fragrant fish pseudomonas vaccine and a preparation method thereof.
(S1) a preparation method of a pseudomonas for killing the fragrant fish (preservation number is CGMCC No. 8985) inactivating liquid: taking out original bacterial liquid from-80 ℃, streaking on a TSA plate, picking out light yellow monoclone after 30 hours, culturing at 30 ℃ for 12 hours, inoculating to 50mLTSB liquid culture medium according to 1% (v/v) inoculum size, culturing at 30 ℃ for 14 hours, adding formaldehyde solution until the final concentration of formaldehyde is 0.5% (v/v), inactivating at 28 ℃ for 48 hours, and respectively taking 0.1mL of TSA coated plate and LB plate. The next day the plate showed no bacterial growth, indicating complete inactivation, and the OD was measured by a spectrophotometer 600 Diluting with sterilized physiological seawater to a concentration of 8X10 7 And (5) CFU/mL for standby to obtain the pseudomonas killing liquid.
(S2) preparing chitosan oligosaccharide (purchased from Qingdao Bozhi Hui Living organism Co., ltd.) into 2mg/mL chitosan solution by using sterilized physiological seawater, and filtering and sterilizing the chitosan oligosaccharide solution with a 0.22 μm filter membrane for later use; then uniformly mixing the sterilized chitosan solution with the pseudomonas killing inactivated liquid prepared in the step (S1) to obtain an inactivated pseudomonas killing vaccine;
wherein, the physiological seawater formula is as follows (each component is dissolved in deionized water):
NaCl 20g/L；
MgCl 2 ·6H 2 O 4.8g/L；
MgSO 4 ·7H 2 O 3.5g/L；
KCl 0.7g/L；
NaHCO 3 0.11g/L；
CaCl 2 ·2H 2 O 1.21g/L；
sterilizing at 121deg.C for 20 min.
EXAMPLE 2 immunopotentiation studies of Chitosan oligosaccharide alone
Sterilized physiological seawater was used as a Control group (fig. 1, "Control");
the chitosan oligosaccharide solution (filtered and sterilized by a filter membrane of 0.22 μm) with the concentration of 1mg/mL prepared by using physiological seawater is taken as an immune group (COS in figure 1);
each group of 20 large yellow croakers is subjected to intraperitoneal injection of 0.1mL of a control group or an immune group, immunization is carried out for 1 month, the wild fragrant fish pseudomonas XSDHY-P is subjected to intraperitoneal injection for toxicity elimination, and the accumulated death condition is observed 14 days after the toxicity elimination, so that the result is shown in figure 1.
As can be seen from FIG. 1, the immune group has a slightly lower mortality rate than the control group, and shows a certain immune enhancing effect, but the enhancing effect has weak resistance to wild Vibrio anguillarum, and the immunity protection is about 10%.
Example 3 injection of immune zebra fish
The immunization groups were set as follows:
(1) Pseudomonas killing liquid group (P.plecloglossica): 8X 10 7 Mixing CFU/mL of the pseudomonas killing liquid for killing the fragrant fish with physiological seawater in an equal volume;
(2) Group of Pseudomonas killing fish inactivating liquid+Chitosan oligosaccharide (P.pleoglossiciida+10 mg/mL COS): 8X 10 7 Mixing CFU/mL of the pseudomonas killing liquid with the chitosan oligosaccharide solution of 20mg/mL in an equal volume;
(3) Group of Pseudomonas killing fish inactivating liquid+Chitosan oligosaccharide (P.pleoglossiciida+1 mg/mL COS): 8X 10 7 Mixing CFU/mL of the pseudomonas killing liquid with 2mg/mL of chitosan oligosaccharide solution in an equal volume;
(4) Pseudomonas killing fish inactivating liquid+chitosan oligosaccharide (P.pleoglossiciida+0.1 mg/mL COS group): 8X 10 7 Mixing CFU/mL of the pseudomonas killing liquid with 0.2mg/mL of chitosan oligosaccharide solution in an equal volume;
the chitosan oligosaccharide solution is obtained by diluting with physiological seawater, and then filtering and sterilizing with a 0.22 μm filter membrane for later use.
Control group: taking sterilized physiological seawater as a control group;
the immune group and the control group each had 30 tails, and 3 parallel groups were set.
The same 5 mu L/tail of zebra fish is injected into the immune groups, and the dosage of the pseudomonas inactivated liquid for killing the fragrant fish in each immune group is 2 multiplied by 10 5 CFU/tail, wild-type one month after immunizationThe strain of Pseudomonas fragi XSDHY-P attacks the virus, and the cumulative death condition of two weeks after the attack is observed, and the immunoprotection (RPS) of each immune group is calculated.
Rps= (1-immune group cumulative mortality/control group cumulative mortality) ×100%
The results are shown in FIG. 2. As can be seen from fig. 2, the pseudomonas killing inactivated liquid is used as an antigen, and chitosan oligosaccharide is added as an immunoadjuvant, so that the chitosan oligosaccharide has a good immunity promoting effect, and the death rate is remarkably reduced. Both 0.1mg/mL and 10mg/mL have remarkable effects; wherein, the addition amount of the chitosan oligosaccharide is optimal at 1mg/mL, which is obviously better than the cases of 0.1mg/mL and 10mg/mL.
Example 4 injection immunization of Large yellow croaker
The immunization groups were set as follows:
(1) Pseudomonas killing liquid group (P.plecloglossica): diluting the Pseudomonas killing liquid with physiological seawater to 2×10 8 CFU/mL (40 mL), 0.1 mL/tail intraperitoneal injection of large yellow croaker, 2X 10 7 CFU/tail.
(2) Group of pseudomonas inactivated liquid + chitosan oligosaccharide (p.pleoglossiciida + COS): diluting the Pseudomonas killing liquid with physiological seawater to 4×10 8 CFU/mL (20 mL), the using amount of chitosan oligosaccharide on large yellow croaker is tentatively 100 mug/tail, namely 0.1mL of chitosan oligosaccharide with the final concentration of 1mg/mL is injected, 2mg/mL (20 mL) of chitosan oligosaccharide is prepared by using physiological seawater, filtered and sterilized by a filter membrane with the thickness of 0.22 mu m, and then the chitosan oligosaccharide and the physiological seawater are mixed in equal volume.
Control group: 0.1 mL/tail abdominal cavity of physiological seawater.
1) Immunoprotection assay
70 animals of each of the experimental group and the control group are respectively provided, three parallel groups are set, and the dosage of the immune group pseudomonas killing liquid is 2 multiplied by 10 7 CFU/tail. The wild strain Pseudomonas fragi XSDHY-P was used for intraperitoneal injection to combat viruses 4, 8 and 12 weeks after immunization, and the cumulative death was observed for two weeks to calculate the immunoprotection (RPS) of each immune group, as shown in FIG. 3. Therefore, the pseudomonas killing inactivated liquid for the fragrant fish is taken as an antigen, and chitosan oligosaccharide is added, so that the pseudomonas killing inactivated liquid has a good immunity promoting effect, and the death rate is obviously reduced.
2) Antibody titers and cross-immune assays
Serum from 4, 8 and 12 weeks after immunization of each immunized group and control group was taken, and after mixing 3 fish serum from each group at each time point, antibody titer and cross-immunization analysis were performed. The interaction of specific antibodies in serum with P.plectogossiciida (CGMCC No. 8985), aeromonas salmonicida A.salmonolide (ATCC 33658) and Aeromonas sulbert A.schubertiii (ATCC 43700) was analyzed mainly using enzyme-linked immunosorbent assay (ELISA) "sandwich method".
The serum to be tested was diluted 10-fold and each group was diluted 1X 10 8 CFU/mL Pseudomonas fragi XSDHY-P, aeromonas salmonicida A.salmonolide, and Aeromonas sulbert A.schubertiii were wrapped.
a. Wrapping overnight: cultivation of A.salmoniced, A.schubertiii and XSDHY-P, three strains were diluted to 1X 10 in physiological seawater, respectively 8 CFU/ml, 100. Mu.L/well were incubated overnight at 4℃in 96-well plates, respectively, coated with A.salmoniced, A.schubertiii, and XSDHY-P.
Pbsta blocking: the liquid in the 96-well plate was dried by pipetting, adding 300. Mu.LPBST (PBS+0.05% Tween-20) to each well, standing for 3min, discarding the liquid, pipetting the 96-well plate, repeating the operation 3 times, adding 100. Mu.L of blocking solution PBSTA (PBST+1% BSA) to each well, and blocking at 22℃for 2h. The blocking solution was discarded, washed 3 times with wash solution PBST, and the well plate was patted dry.
c. Adding serum for incubation: serum of the experimental group and the control group to be tested (the serum is properly diluted by PBST according to actual conditions, and is generally diluted by about 10 times), 100 mu L/hole, two compound holes and blank control are added with PBST, after incubation is carried out for 3 hours at 22 ℃, the 96-well plate is patted dry, washed 5 times by PBST, washed last time and stood for 5min, and then patted dry.
d. Adding an antibody for incubation: mu.L of murine antigen Large yellow croakert IgM (purchased from Aquatic DiagnosticsLtd) was added to each well, incubated at 22℃for 1h, washed 5 times with PBST, washed the last time, left for 5min and then patted dry.
e. Adding secondary antibody for incubation: 100 μl of goat anti-mouse IgG-HRP (ex Abigen) was added to each well, incubated at 22 ℃ for 1h, washed 5 times with PBST, washed the last time, left for 5min and then patted dry.
TMB substrate development: 100. Mu.L of soluble TMB substrate (Tiangen biochemical reagent) was added to each well, and the wells were developed for 10min at room temperature in the dark, at which time the positive wells developed blue.
g. The color development was terminated and read: 50 mu L of each well was charged with 2mol/L H 2 SO 4 Terminating the reaction, wherein the positive holes are yellow, and measuring the OD of each hole by using an enzyme-labeling instrument 450 Is a reading of (a).
The results are shown in FIGS. 4-6. The result shows that the inactivated liquid for killing the pseudomonas of the fish is taken as an antigen, and the chitosan oligosaccharide is added, so that the immune effect is obvious, the serum antibody level of the yellow croaker after the immune is obviously improved, and the inactivated liquid has a certain cross immune protection effect on the pseudomonas salmonicida and the pseudomonas sulbert.
Example 5 adjuvant residual experiment
The immunization groups were set as follows:
1) Pseudomonas killing liquid group (P.plecloglossica): diluting the Pseudomonas killing liquid with physiological seawater to 2×10 8 CFU/mL (40 mL), 0.1 mL/tail intraperitoneal injection of large yellow croaker, 2X 10 7 CFU/tail.
2) Pseudomonas killing liquid + chitosan oligosaccharide group (P.pleoglossiclida + COS): diluting the Pseudomonas killing liquid with physiological seawater to 4×10 8 CFU/mL (20 mL), the using amount of chitosan oligosaccharide on large yellow croaker is tentatively 100 mug/tail, namely 0.1mL of chitosan oligosaccharide with the final concentration of 1mg/mL is injected, 2mg/mL (20 mL) of chitosan oligosaccharide is prepared by using physiological seawater, filtered and sterilized by a filter membrane with the thickness of 0.22 mu m, and then the chitosan oligosaccharide and the physiological seawater are mixed in equal volume.
3) Pseudomonas killing liquid for killing fragrant fish + MONTANIDE TM ISA763A adjuvant group (p.pleoglossiciida+763): diluting the Pseudomonas killing liquid with physiological seawater to 6×10 8 CFU/mL (12 mL), 763 adjuvant (28 mL) 3:7, and the final concentration of the Pseudomonas fragi killing liquid is 2×10 8 CFU/mL (40 mL). 0.1 mL/tail intraperitoneal injection of immune large yellow croaker, namely the immune dose of the pseudomonas killing liquid for killing the fragrant fish is 2 multiplied by 10 7 CFU/tail.
The control group was set as follows:
1) Physiological seawater group (Control): sterilizing the prepared physiological seawater at 121 ℃ for 20 minutes for later use.
2) Chitosan oligosaccharide group alone (COS): the chitosan oligosaccharide with the concentration of 1mg/mL is prepared by using physiological seawater, and is filtered and sterilized by a filter membrane with the concentration of 0.22 mu m for standby.
3) 763 groups alone (763): sterilized physiological seawater was mixed with 763 oil adjuvant 3:7, uniformly mixing and emulsifying for later use.
Each immune group and the control group were injected with 0.1 mL/tail abdominal cavity of large yellow croaker (20/group), three months after immunization, and the residual condition of the adjuvant in each immune group and the viscera of the control group was anatomically observed.
As a result, it was found that the viscera of the yellow croaker in the immune group and the control group, particularly the liver, which had been engaged with 763 oil adjuvant, were covered or coated with a layer of milky flocculent compound, that is, 763 adjuvant remained, and the viscera of the other immune group and the control group were clean and free of impurities, as shown in fig. 7.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the explanation of the present invention, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. An inactivated pseudomonas vaccine for killing fish, which is characterized by comprising an inactivated pseudomonas liquid for killing the fish and chitosan oligosaccharide;
in the vaccine, the concentration of the pseudomonas killing liquid for killing the fragrant fish is 5 multiplied by 10 6 CFU/mL～3×10 8 CFU/mL, the concentration of chitosan oligosaccharide is 0.05 mg/mL-50 mg/mL.
2. An inactivated pseudomonas fish vaccine according to claim 1, wherein the concentration of pseudomonas fish inactivated solution in the vaccine is 1 x 10 7 CFU/mL～1.5×10 8 CFU/mThe concentration of the L, chitosan oligosaccharide is 0.1 mg/mL-10 mg/mL.
3. An inactivated pseudomonas fish vaccine according to claim 1, wherein the concentration of pseudomonas fish inactivating liquid in the vaccine is 2 x 10 7 CFU/mL～8×10 7 CFU/mL, the final concentration of chitosan oligosaccharide is 0.5 mg/mL-5 mg/mL.
4. An inactivated vaccine against pseudomonas for fragrant fishes according to claim 1, wherein the chitosan oligosaccharide has an average molecular weight of 2000 and a degree of deacetylation higher than 90%;
the preservation number of the pseudomonas for killing the fragrant fish is CGMCC No.8985.
5. A method of preparing an inactivated vaccine against pseudomonas for aromaticalis according to any one of claims 1 to 4, comprising the steps of:
(S1) inoculating activated pseudomonas for killing the fragrant fish into a liquid culture medium for pre-culturing, and then adding formaldehyde for inactivating treatment to obtain pseudomonas for killing the fragrant fish inactivating liquid;
(S2) after the post-treatment of the pseudomonas putida inactivating liquid prepared in the step (S1) is uniformly mixed with the chitosan oligosaccharide solution, so as to obtain the pseudomonas putida inactivating vaccine.
6. The method of claim 5, wherein in step (S1), the liquid medium is TSB liquid medium, and the formaldehyde concentration is less than 0.5% by volume.
7. The method for preparing an inactivated vaccine against pseudomonas for fish according to claim 5, wherein, in step (S1), the temperature is 30 ℃ and the time is 14 hours during the pre-culture;
the post-treatment is dilution with physiological seawater;
in the inactivation treatment process, the temperature is 28 ℃, the time is 48 hours, and the bacterial liquid is fully stirred for 1 time every 6 hours.
8. The method for preparing an inactivated vaccine against pseudomonas fish according to claim 7, wherein the physiological seawater comprises NaCl, mgCl 2 ·6H 2 O、MgSO 4 ·7H 2 O、KCl、NaHCO 3 、CaCl 2 ·2H 2 O；
Wherein the concentration of NaCl is 20g/L, mgCl 2 ·6H 2 The concentration of O is 4.8g/L, mgSO 4 ·7H 2 The concentration of O is 3.5g/L, the concentration of KCl is 0.7g/L, and the concentration of NaHCO is 0.7g/L 3 At a concentration of 0.11g/L CaCl 2 ·2H 2 The concentration of O was 1.21g/L.
9. Use of an inactivated vaccine against pseudomonas fish according to any one of claims 1 to 4 in the preparation of a composition for preventing pathogenic bacterial infection in fish.
10. A composition for preventing pathogenic bacterial infection in fish, comprising the inactivated pseudomonas fragi-cidal vaccine of any one of claims 1 to 4;
the pathogenic bacteria are selected from one or more of Pseudomonas fragi, aeromonas salmonicida or Aeromonas sulunder.
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