CN112807426A - Polymer adjuvant based on animal vaccine, preparation method and application thereof - Google Patents
Polymer adjuvant based on animal vaccine, preparation method and application thereof Download PDFInfo
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- 239000002671 adjuvant Substances 0.000 title claims abstract description 57
- 229960005486 vaccine Drugs 0.000 title claims abstract description 57
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- 238000002360 preparation method Methods 0.000 title abstract description 5
- 238000002347 injection Methods 0.000 claims abstract description 46
- 239000007924 injection Substances 0.000 claims abstract description 46
- 229920005610 lignin Polymers 0.000 claims abstract description 46
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- 241000588832 Bordetella pertussis Species 0.000 claims abstract description 33
- XETCRXVKJHBPMK-MJSODCSWSA-N trehalose 6,6'-dimycolate Chemical compound C([C@@H]1[C@H]([C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](COC(=O)C(CCCCCCCCCCC3C(C3)CCCCCCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)O2)O)O1)O)OC(=O)C(C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC XETCRXVKJHBPMK-MJSODCSWSA-N 0.000 claims abstract description 33
- 241000405911 Rehmannia glutinosa Species 0.000 claims abstract description 32
- 239000010404 Scutellaria baicalensis extract Substances 0.000 claims abstract description 32
- 239000001599 crocus sativus l. flower extract Substances 0.000 claims abstract description 32
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- 239000002253 acid Substances 0.000 claims abstract description 21
- 239000007864 aqueous solution Substances 0.000 claims abstract description 21
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 10
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 6
- WERKSKAQRVDLDW-ANOHMWSOSA-N [(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO WERKSKAQRVDLDW-ANOHMWSOSA-N 0.000 claims description 6
- 229950004959 sorbitan oleate Drugs 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 4
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- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 2
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- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 1
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- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
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- 102000004127 Cytokines Human genes 0.000 description 1
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
-
- 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
- A61K2039/55588—Adjuvants of undefined constitution
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Inorganic Chemistry (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention is applicable to the field of animal vaccines, and provides a polymer adjuvant based on animal vaccines, a preparation method and application thereof, wherein the polymer adjuvant based on animal vaccines comprises the following raw materials in parts by weight: 38-49 parts of an aqueous solution for injection, 55-75 parts of vegetable oil for injection, 12-19 parts of a compound polymer, 5-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-8 parts of trehalose dimycolate, 2-5 parts of polyinosinic acid-polycytidylic acid, 2-5 parts of bordetella pertussis, 8-18 parts of a scutellaria baicalensis extract, 2-5 parts of a saffron extract and 1-4 parts of a rehmannia glutinosa extract; the animal vaccine-based polymer adjuvant provided by the embodiment of the invention is compounded by the modified lignin and the modified polyacrylic copolymer according to a proportion and then added into a system to play a role in synergy, and the prepared adjuvant is stable and efficient, and is worthy of popularization and application.
Description
Technical Field
The invention belongs to the field of animal vaccines, and particularly relates to a polymer adjuvant based on an animal vaccine, a preparation method and an application thereof.
Background
An adjuvant is a substance that is applied simultaneously or in advance with an antigen, and that enhances the body's ability to respond to the antigen or alters the type of immune response. Adjuvant plays a crucial role for vaccine, it can act on antigen and organism at the same time, can regulate and even change the type of humoral and/or cellular immune response that the organism immune system produces antigen specificity, the mechanism of action of adjuvant is identical to immune response process of organism, activate antigen presenting cell first, intake foreign antigen, process and present, then further activate T cell and B cell, through the fine regulation of cytokine network, adjuvant composition can be designed to be favorable to strengthening Th1 or Th2 immune response to vaccine antigen.
The benefit of adding adjuvants to vaccine formulations to enhance the immunogenicity of the vaccine must be balanced against the risk of causing adverse reactions. With the development of immunological technology research, people realize that the selection of a proper adjuvant becomes the key for enhancing the immune effect, the effect of improving the response intensity of the antigen, the immune maintenance time, the immune tolerance and the like is limited by singly using the antigen or only using one adjuvant, and the vaccine using the compound adjuvant can induce stronger immune response than the vaccine using a single adjuvant, so that the compound immune adjuvant prepared by matching different types of adjuvants becomes a new trend of adjuvant development
The existing adjuvant rarely uses the component compounding way of vegetable oil, polymer and the like to prepare the animal vaccine adjuvant, so that the development of the component compounding way of the polymer, the vegetable oil and the like to prepare the stable and efficient vaccine adjuvant is still valuable.
Disclosure of Invention
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention is realized in such a way that the polymer adjuvant based on the animal vaccine comprises the following raw materials in parts by weight: 38-49 parts of an aqueous solution for injection, 55-75 parts of vegetable oil for injection, 12-19 parts of a compound polymer, 5-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-8 parts of trehalose dimycolate, 2-5 parts of polyinosinic acid-polycytidylic acid, 2-5 parts of bordetella pertussis, 8-18 parts of a scutellaria baicalensis extract, 2-5 parts of a saffron extract and 1-4 parts of a rehmannia glutinosa extract;
the compound polymer is prepared from modified lignin and a modified polyacrylic acid copolymer according to the weight ratio of 1: (1.5-3.2).
As a further scheme of the invention: the modified lignin is prepared by the following steps: mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 4-18h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 8-60 g/L; the dosage of the biocatalyst is 100-100000U/L.
As a further scheme of the invention: the modified polyacrylic acid copolymer is prepared by the following steps: dissolving levo-pinitol, polyacrylic acid and a catalyst in a mixed solution of chloroform and tetrahydrofuran, adding ethyl-3-carbodiimide hydrochloride to perform sealing reaction for 1h under the nitrogen environment and at the temperature of 10 ℃, heating to 40 ℃, performing reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain the modified polyacrylic acid copolymer.
As a further scheme of the invention: the catalyst is selected from 4-dimethylamino pyridine.
As a further scheme of the invention: the vegetable oil for injection is selected from one of soybean oil and peanut oil; the emulsifier is selected from one of sorbitol monooleate, sorbitol monolaurate, sorbitan oleate and sucrose oleate; the auxiliary agent is a straight chain or branched chain fatty acid or fatty alcohol with a carbon chain length of 3-22.
As a further scheme of the invention: the feed comprises the following raw materials in parts by weight: 40-47 parts of an aqueous solution for injection, 58-72 parts of vegetable oil for injection, 14-17 parts of a compound polymer, 6-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-7 parts of trehalose dimycolate, 2-4 parts of polyinosinic acid-polycytidylic acid, 2-4 parts of bordetella pertussis, 8-15 parts of a scutellaria baicalensis extract, 2-4 parts of a saffron extract and 1-3 parts of a rehmannia glutinosa extract.
As a further scheme of the invention: the feed comprises the following raw materials in parts by weight: 43-45 parts of water solution for injection, 60-68 parts of vegetable oil for injection, 15-16 parts of compound polymer, 6-7 parts of emulsifier, 1-2 parts of auxiliary agent, 5-7 parts of trehalose dimycolate, 3-4 parts of polyinosinic acid-polycytidylic acid, 3-4 parts of bordetella pertussis, 10-13 parts of scutellaria baicalensis extract, 3-4 parts of saffron extract and 2-3 parts of rehmannia glutinosa extract.
As a further scheme of the invention: the feed comprises the following raw materials in parts by weight: 44 parts of water solution for injection, 65 parts of vegetable oil for injection, 15 parts of compound polymer, 6 parts of emulsifier, 2 parts of auxiliary agent, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of scutellaria baicalensis extract, 3 parts of saffron extract and 2 parts of rehmannia glutinosa extract.
A polymer adjuvant based on animal vaccine, a preparation method and application thereof comprise the following steps:
1) taking the following raw materials in parts by weight: 38-49 parts of an aqueous solution for injection, 55-75 parts of vegetable oil for injection, 12-19 parts of a compound polymer, 5-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-8 parts of trehalose dimycolate, 2-5 parts of polyinosinic acid-polycytidylic acid, 2-5 parts of bordetella pertussis, 8-18 parts of a scutellaria baicalensis extract, 2-5 parts of a saffron extract and 1-4 parts of a rehmannia glutinosa extract;
2) adding an emulsifier and an auxiliary agent into vegetable oil for injection, and physically mixing to obtain emulsion A;
3) adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B;
4) sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C;
5) and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 3-6h at 40-80 ℃, and controlling the stirring speed to be below 3000 r/min to obtain the emulsion.
An application of a polymer adjuvant based on an animal vaccine in the field of animal vaccines.
The animal vaccine-based polymer adjuvant provided by the embodiment of the invention is compounded by the modified lignin and the modified polyacrylic copolymer according to a proportion and then added into a system to play a role in synergy, and the prepared adjuvant is stable and efficient, and is worthy of popularization and application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The technical effects of the polymer adjuvant based on animal vaccine of the present invention will be further described with reference to the following specific examples, but the specific implementation methods mentioned in these examples are only illustrative and explanatory of the technical scheme of the present invention, and do not limit the implementation scope of the present invention, and all modifications and substitutions based on the above principles should be within the protection scope of the present invention.
Example 1
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 49 parts of an injection water solution, 75 parts of soybean oil, 19 parts of a compound polymer, 9 parts of sorbitol monooleate, 3 parts of branched fatty alcohol with a carbon chain length of 3-22, 8 parts of trehalose dimycolate, 5 parts of polyinosinic acid-polycytidylic acid, 5 parts of bordetella pertussis, 18 parts of a scutellaria baicalensis extract, 5 parts of a saffron extract and 4 parts of a rehmannia glutinosa extract; adding sorbitol monooleate and branched fatty alcohol with a carbon chain length of 3-22 into soybean oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 6 hours at 40 ℃, and controlling the stirring speed to 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 2
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 38 parts of an aqueous solution for injection, 55 parts of soybean oil, 12 parts of a compound polymer, 5 parts of sorbitol monooleate, 1 part of branched fatty alcohol with a carbon chain length of 3-22, 2 parts of trehalose dimycolate, 2 parts of polyinosinic acid-polycytidylic acid, 2 parts of bordetella pertussis, 8 parts of a scutellaria baicalensis extract, 2 parts of a saffron extract and 1 part of a rehmannia glutinosa extract; adding sorbitol monooleate and branched fatty alcohol with a carbon chain length of 3-22 into soybean oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 3h at 80 ℃, and controlling the stirring speed to 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 3
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 47 parts of an injection water solution, 72 parts of soybean oil, 17 parts of a compound polymer, 9 parts of sorbitol monolaurate, 3 parts of straight-chain fatty alcohol with a carbon chain length of 3-22, 7 parts of trehalose dimycolate, 4 parts of polyinosinic acid-polycytidylic acid, 4 parts of bordetella pertussis, 15 parts of a scutellaria baicalensis extract, 4 parts of a saffron extract and 3 parts of a rehmannia glutinosa extract; adding sorbitol monolaurate and straight-chain fatty alcohol with a carbon chain length of 3-22 into soybean oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 4
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 40 parts of an aqueous solution for injection, 58 parts of soybean oil, 14 parts of a compound polymer, 6 parts of sorbitan oleate, 1 part of straight-chain fatty alcohol with a carbon chain length of 3-22, 2 parts of trehalose dimycolate, 2 parts of polyinosinic acid-polycytidylic acid, 2 parts of bordetella pertussis, 8 parts of a scutellaria baicalensis extract, 2 parts of a saffron extract and 1 part of a rehmannia glutinosa extract; adding sorbitan oleate and straight-chain fatty alcohol with the carbon chain length of 3-22 into soybean oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 5
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 45 parts of water solution for injection, 68 parts of peanut oil, 16 parts of compound polymer, 7 parts of sorbitan oleate, 2 parts of branched fatty alcohol, 7 parts of trehalose dimycolate, 4 parts of polyinosinic acid-polycytidylic acid, 4 parts of bordetella pertussis, 13 parts of scutellaria baicalensis extract, 4 parts of saffron extract and 3 parts of rehmannia glutinosa extract; adding sorbitan oleate and branched fatty alcohol into peanut oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 6
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 43 parts of water solution for injection, 60 parts of peanut oil, 15 parts of compound polymer, 6 parts of sucrose oleate, 1 part of branched fatty alcohol, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of scutellaria baicalensis extract, 3 parts of saffron extract and 2 parts of rehmannia glutinosa extract; adding sucrose oleate and branched fatty alcohol into peanut oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Example 7
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; the modified lignin and the modified polyacrylic acid copolymer are prepared according to the following weight ratio of 1: 2 preparing a compound polymer for later use; taking the following raw materials in parts by weight: 44 parts of an injection water solution, 65 parts of peanut oil, 15 parts of a compound polymer, 6 parts of sucrose oleate, 2 parts of branched fatty acid, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of a scutellaria baicalensis extract, 3 parts of a saffron extract and 2 parts of a rehmannia glutinosa extract; adding sucrose oleate and fatty acid with branched chain into peanut oil, and physically mixing to obtain emulsion A; adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Comparative example 1
Taking the following raw materials in parts by weight: 44 parts of water solution for injection, 65 parts of peanut oil, 6 parts of sucrose oleate, 2 parts of branched fatty acid, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of scutellaria baicalensis extract, 3 parts of saffron extract and 2 parts of rehmannia glutinosa extract; adding sucrose oleate and fatty acid with branched chain into peanut oil, and physically mixing to obtain emulsion A; mixing trehalose dimycolate, polyinosinic-polycytidylic acid, Bordetella pertussis, Scutellariae radix extract, stigma croci Sativi extract and rehmanniae radix extract uniformly to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Comparative example 2
Mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 12h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 40 g/L; the using amount of the biological 4-dimethylaminopyridine is 500U/L, and modified lignin is prepared for later use; taking the following raw materials in parts by weight: 44 parts of an injection water solution, 65 parts of peanut oil, 15 parts of modified lignin, 6 parts of sucrose oleate, 2 parts of branched fatty acid, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of a scutellaria baicalensis extract, 3 parts of a saffron extract and 2 parts of a rehmannia glutinosa extract; adding sucrose oleate and fatty acid with branched chain into peanut oil, and physically mixing to obtain emulsion A; adding the modified lignin into an aqueous solution for injection, and physically mixing to obtain an emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Comparative example 3
Dissolving levo-pinitol, polyacrylic acid and 4-dimethylaminopyridine in a mixed solution of chloroform and tetrahydrofuran, controlling the temperature at 10 ℃ in a nitrogen environment, adding ethyl-3-carbodiimide hydrochloride, carrying out sealing reaction for 1h, heating to 40 ℃, carrying out reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain a modified polyacrylic acid copolymer for later use; taking the following raw materials in parts by weight: 44 parts of an injection water solution, 65 parts of peanut oil, 15 parts of modified polyacrylic acid copolymer, 6 parts of sucrose oleate, 2 parts of branched fatty acid, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of scutellaria baicalensis extract, 3 parts of saffron extract and 2 parts of rehmannia glutinosa extract; adding sucrose oleate and fatty acid with branched chain into peanut oil, and physically mixing to obtain emulsion A; adding the modified polyacrylic acid copolymer into an aqueous solution for injection, and physically mixing to obtain emulsion B; sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C; and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 5 hours at the temperature of 60 ℃, and controlling the stirring speed to be less than 2800 revolutions per minute to obtain the polymer adjuvant based on the animal vaccine.
Experimental group
Sterilizing the polymer adjuvants based on the animal vaccines of examples 1-7 and comparative examples 1-3 at 121 ℃ for 15 minutes, cooling to 25 ℃, adding 500ml of the polymer adjuvants into a vaccine mixing tank, slowly adding 500ml of the porcine circovirus antigen meeting the production standard into the mixing tank under the condition of controlling the stirring rate at 2000 rpm, stirring and mixing for 5 minutes to prepare the porcine circovirus vaccine, and detecting after subpackaging and marking. After 24 hours of standing, physical index detection and animal experiments are carried out.
Examples of the experiments
The experimental example is used for explaining index detection of the animal vaccine containing the polymer combined adjuvant of the invention, and the relevant description is as follows:
1. stability test
The experimental group porcine circular vaccines prepared by the methods of examples 1-7 and comparative examples 1-3 are stable when placed at 2-8 ℃ for 2 years.
2. Sterility testing
10g of casein peptone, 1000ml of meat extract, 5g of sodium chloride and 15-20 g of agar, adding the casein peptone and the sodium chloride into the meat extract, dissolving at a low temperature, adjusting the pH value to be alkalescent, boiling, filtering, adjusting the pH value to be 7.2 +/-0.2 after sterilization, subpackaging and sterilizing. After the prepared experimental group vaccine and the culture medium are inoculated, the experimental group vaccine is cultured for 48 hours at the temperature of 30-35 ℃, and the observation results are all aseptic.
3. Animal immunization evaluation
The evaluation contents are as follows: after the animal is inoculated with the vaccine, no abnormal reaction occurs, blood is collected at a specified time, serum is separated, and the serum antibody titer is detected.
Experimental groups: porcine circular vaccines of examples 1-7 of the present invention and comparative examples 1-3;
blank control group: no vaccine was injected.
a. Animal safety test
1. 2 guinea pigs weighing 350-450 g were injected subcutaneously with 2ml of vaccine per animal: 5 mice weighing 18-22 g were injected subcutaneously with 0.5ml of vaccine per mouse. The observation was continued for 7 days to observe clinical response. After continuously observing for 7 days, no abnormal clinical reaction appears in all the observations.
b. Antibody detection
The experimental method comprises the following steps:
animal immunization was performed as per protocol requirements: 110 PCV2ELISA antibody negative and healthy female clean-grade Balb/c (PCV2ELISA antibody titer is not higher than 1:50) are used for dividing into 11 groups, 10 groups are used, and 1-10 groups are respectively subcutaneously inoculated with the experimental group pig circular ring polymer combined adjuvant vaccine, each 0.2 ml, and the 11 th group is not inoculated, and the blank control is adopted. Each group of mice was kept separately for observation. Blood was collected at 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks after immunization, and serum was separated to determine PCV2ELISA antibody titer in the serum. The average antibody titer of the polymer combined adjuvant groups provided by the examples 1-7 of the invention is higher than that of the polymer combined adjuvant groups provided by the comparative examples 1-3, and higher antibodies appear in one week after immunization. Specific data are shown in table 1.
TABLE 1
| Item | 1 week (not less) | 2 weeks (not less) | 3 weeks (not less) | 4 weeks (not less) | 5 weeks (not less) |
| Example 1 | 1：500 | 1：1100 | 1：2000 | 1：3800 | 1：3800 |
| Example 2 | 1：500 | 1：1000 | 1：2000 | 1：3800 | 1：3800 |
| Example 3 | 1：500 | 1：1000 | 1：2000 | 1：3800 | 1：3800 |
| Example 4 | 1：500 | 1：1100 | 1：2000 | 1：3800 | 1：3800 |
| Example 5 | 1：500 | 1：1100 | 1：2000 | 1：3800 | 1：3800 |
| Example 6 | 1：500 | 1：1000 | 1：2000 | 1：3800 | 1：3800 |
| Example 7 | 1：500 | 1：1100 | 1：2100 | 1：4000 | 1：4000 |
| Comparative example 1 | 1：150 | 1：300 | 1：600 | 1：1000 | 1：1000 |
| Comparative example 2 | 1：150 | 1：300 | 1：600 | 1：1000 | 1：1000 |
| Comparative example 3 | ≤1:50 | 1:50 | 1:100 | 1:100 | 1:100 |
| Blank control | ≤1:50 | ≤1:50 | ≤1:50 | ≤1:50 | ≤1:50 |
The results show that the polymer adjuvant based on the animal vaccine prepared in the embodiments 1-7 of the invention is stable and efficient, compared with the polymer adjuvant based on the animal vaccine prepared in the comparative examples 1-3, the addition of the compound polymer in the embodiments improves the performance of the adjuvant, and compared with the independent modified lignin and the modified polyacrylic acid copolymer, the modified lignin and the modified polyacrylic acid copolymer are compounded and added into the system according to the proportion to play a role in synergy.
In conclusion, the animal vaccine-based polymer adjuvant provided by the embodiment of the invention is compounded by the modified lignin and the modified polyacrylic acid copolymer according to a proportion and then added into a system to play a role in synergy, and the prepared adjuvant is stable and efficient, and is worthy of popularization and application.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The polymer adjuvant based on the animal vaccine is characterized by comprising the following raw materials in parts by weight: 38-49 parts of an aqueous solution for injection, 55-75 parts of vegetable oil for injection, 12-19 parts of a compound polymer, 5-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-8 parts of trehalose dimycolate, 2-5 parts of polyinosinic acid-polycytidylic acid, 2-5 parts of bordetella pertussis, 8-18 parts of a scutellaria baicalensis extract, 2-5 parts of a saffron extract and 1-4 parts of a rehmannia glutinosa extract;
the compound polymer is prepared from modified lignin and a modified polyacrylic acid copolymer according to the weight ratio of 1: (1.5-3.2).
2. The animal vaccine-based polymeric adjuvant of claim 1, wherein the modified lignin is produced by: mixing lignin and phenol monooxygenase in a phosphate buffer solution, and reacting at room temperature for 4-18h to obtain the lignin-phenol monooxygenase aqueous solution; the concentration of the lignin is 8-60 g/L; the dosage of the biocatalyst is 100-100000U/L.
3. The animal vaccine-based polymeric adjuvant of claim 1, wherein the modified polyacrylic acid copolymer is prepared by: dissolving levo-pinitol, polyacrylic acid and a catalyst in a mixed solution of chloroform and tetrahydrofuran, adding ethyl-3-carbodiimide hydrochloride to perform sealing reaction for 1h under the nitrogen environment and at the temperature of 10 ℃, heating to 40 ℃, performing reaction for 72h, removing the solvent after the reaction is finished, and dialyzing with absolute ethyl alcohol to obtain the modified polyacrylic acid copolymer.
4. The animal vaccine-based polymer adjuvant of claim 3, wherein the catalyst is selected from the group consisting of 4-dimethylaminopyridine.
5. The animal vaccine-based polymeric adjuvant of claim 1, wherein the injectable vegetable oil is selected from one of soybean oil, peanut oil; the emulsifier is selected from one of sorbitol monooleate, sorbitol monolaurate, sorbitan oleate and sucrose oleate; the auxiliary agent is a straight chain or branched chain fatty acid or fatty alcohol with a carbon chain length of 3-22.
6. The animal vaccine-based polymeric adjuvant according to claim 1, comprising the following raw materials in parts by weight: 40-47 parts of an aqueous solution for injection, 58-72 parts of vegetable oil for injection, 14-17 parts of a compound polymer, 6-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-7 parts of trehalose dimycolate, 2-4 parts of polyinosinic acid-polycytidylic acid, 2-4 parts of bordetella pertussis, 8-15 parts of a scutellaria baicalensis extract, 2-4 parts of a saffron extract and 1-3 parts of a rehmannia glutinosa extract.
7. The animal vaccine-based polymeric adjuvant according to claim 1, comprising the following raw materials in parts by weight: 43-45 parts of water solution for injection, 60-68 parts of vegetable oil for injection, 15-16 parts of compound polymer, 6-7 parts of emulsifier, 1-2 parts of auxiliary agent, 5-7 parts of trehalose dimycolate, 3-4 parts of polyinosinic acid-polycytidylic acid, 3-4 parts of bordetella pertussis, 10-13 parts of scutellaria baicalensis extract, 3-4 parts of saffron extract and 2-3 parts of rehmannia glutinosa extract.
8. The animal vaccine-based polymeric adjuvant according to claim 1, comprising the following raw materials in parts by weight: 44 parts of water solution for injection, 65 parts of vegetable oil for injection, 15 parts of compound polymer, 6 parts of emulsifier, 2 parts of auxiliary agent, 5 parts of trehalose dimycolate, 3 parts of polyinosinic acid-polycytidylic acid, 3 parts of bordetella pertussis, 10 parts of scutellaria baicalensis extract, 3 parts of saffron extract and 2 parts of rehmannia glutinosa extract.
9. The method for preparing an animal vaccine based polymeric adjuvant according to any of claims 1-8, comprising the steps of:
1) taking the following raw materials in parts by weight: 38-49 parts of an aqueous solution for injection, 55-75 parts of vegetable oil for injection, 12-19 parts of a compound polymer, 5-9 parts of an emulsifier, 1-3 parts of an auxiliary agent, 2-8 parts of trehalose dimycolate, 2-5 parts of polyinosinic acid-polycytidylic acid, 2-5 parts of bordetella pertussis, 8-18 parts of a scutellaria baicalensis extract, 2-5 parts of a saffron extract and 1-4 parts of a rehmannia glutinosa extract;
2) adding an emulsifier and an auxiliary agent into vegetable oil for injection, and physically mixing to obtain emulsion A;
3) adding the compound polymer into an injection water solution, and physically mixing to obtain emulsion B;
4) sequentially adding trehalose dimycolate, polyinosinic-polycytidylic acid, bordetella pertussis, scutellaria baicalensis extract, saffron extract and rehmannia glutinosa extract into emulsion B, and uniformly mixing to obtain emulsion C;
5) and (3) uniformly stirring and mixing the emulsion A and the emulsion C, stirring for 3-6h at 40-80 ℃, and controlling the stirring speed to be below 3000 r/min to obtain the emulsion.
10. Use of the animal vaccine-based polymer adjuvant according to any one of claims 1-8 in the field of animal vaccines.
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