CN107802832A - A kind of injection-type inactivated vaccine adjuvant for animals - Google Patents

A kind of injection-type inactivated vaccine adjuvant for animals Download PDF

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CN107802832A
CN107802832A CN201711345593.5A CN201711345593A CN107802832A CN 107802832 A CN107802832 A CN 107802832A CN 201711345593 A CN201711345593 A CN 201711345593A CN 107802832 A CN107802832 A CN 107802832A
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adjuvant
sodium alginate
vaccine
injection
vaccine adjuvant
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魏凯
朱瑞良
孙振红
马丽莉
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Shandong Agricultural University
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Shandong Agricultural University
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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
    • A61K39/39—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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/55583—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to a kind of injection-type inactivated vaccine adjuvant for animals and preparation method thereof, the adjuvant is 28 by mass ratio:98 92 sodium alginate and sterilized water composition, to replace conventional white-oil adjuvant etc.；The beneficial effects of the invention are as follows：The injection-type inactivated vaccine adjuvant preparation technology for animals that is there is provided is simple, cost is low, composition is simple, stable dosage forms, easily storage, is easy to inject, easy to use, action effect is good, no toxicity, developing and utilizingpotentiality in animal husbandry is huge, has broad application prospects.

Description

A kind of injection-type inactivated vaccine adjuvant for animals
Technical field
The present invention relates to animal immune field, and in particular to a kind of injection-type inactivated vaccine adjuvant for animals.
Background technology
Inactivated vaccine typically refers to first cultivate virus or bacterium, with heating or chemical agent (being typically formalin) Inactivated, then with immunologic adjuvant coordinate made of vaccine.Particularly in livestock breeding industry, inactivated vaccine is as the most frequently used Conventional vaccine, because of its good security and longer protection period, it is widely used in the generation to keep off infection.Inactivation epidemic disease for animals When seedling produces, immunologic adjuvant is essential.Clinically conventional inactivated vaccine adjuvant for animals has white-oil adjuvant, aluminium hydroxide Adjuvant, propolis etc., wherein white-oil adjuvant are the most frequently used live vaccine adjuvants, when it acts on mainly effective extension antigen release Between, make antigen continue to stimulate body to produce immune response, and antigenic surface product can be increased, it is thin so as to be advantageous to macrophage Born of the same parents capture antigen.However, its shortcoming is also obvious：1) cost is high, more than per ton ten thousand yuan of the high-quality white oil of commercialization；2) Complex manufacturing is, it is necessary to add a variety of surfactants and the emulsifying device of specialty；3) antigen matches limited, maximum antigen Amount proportioning is 1:1, most viral antigens need concentration to can be only achieved requirement；4) body absorbs slow, and stress reaction is big, easily occurs Tissue damage, production performance decline the even side reaction such as death；5) less stable, especially in summer high temperature and long-time storage When easily there is water oil lamination.Therefore, seeking development of the substitute of white-oil adjuvant for injection-type live vaccine has weight Want meaning.
The content of the invention
The main object of the present invention, which is that, overcomes above-mentioned the deficiencies in the prior art, there is provided a kind of injection-type inactivation epidemic disease for animals Seedling adjuvant and preparation method thereof, the adjuvant are 2-8 by mass ratio:98-92 sodium alginate and sterilized water composition, to replace Conventional white-oil adjuvant；The beneficial effects of the invention are as follows：The injection-type inactivated vaccine adjuvant preparation technology for animals that is there is provided is simple, Cost is low, composition is simple, stable dosage forms, easily storage, is easy to inject, easy to use, action effect is good, no toxicity, is raiseeing Developing and utilizingpotentiality in animal husbandry is huge, has broad application prospects.
Sodium alginate of the present invention is the sodium salt that alginic acid changes into, and is from the sea-tangle or sargassum of brown algae The accessory substance after iodine and mannitol is extracted, its molecule is by beta-D-mannuronic acid (β-D-mannuronic, M) and α-L- Gus Lip river Uronic acid (α-L-guluronic, G) is formed by (1 → 4) key connection, is a kind of natural polysaecharides biopolymer, nothing Poison, there is stability, dissolubility, viscosity and security needed for pharmaceutical preparation auxiliary material, and it is cheap and easily-available, at present in food Industry and field of medicaments are widely applied.Sodium alginate is white or pale yellow powder, and almost odorless, tasteless, is soluble in Water, insoluble in organic solvents such as ethanol, ether, chloroforms.Sodium alginate powder molecular weight distribution is wider, micro- after being added to the water The aquation of intergranular makes the surface of sodium alginate have viscosity, according to molecular size range can be divided into superelevation, height, in and it is low viscous Degree, its stability in solution of the pH between 6-11 is good, pH<Solution will separate out alginic acid, pH when 6>Solution can condense when 11, Sodium alginate soln is the most stable during pH=7.For sodium alginate as a kind of linear polysaccharide, its strand is in Coiling-type in the solution Distribution, has good biocompatibility, and medicine can be controlled to be discharged at specific position with specific speed, plays sustained release Effect, and it is nonirritant to body, absorb complete.These characteristics extremely meet the requirement of vaccine adjuvant.
But sodium alginate is medically prepared into gel or micro- with reactions such as calcium chloride or chitosans more in the prior art Capsule, for containing medicine, antigen, cell, gene, albumen etc., for drug therapy, oral vaccine and tissue repair etc., but It is that there has been no the application as injection vaccine adjuvant.The present invention helps sodium alginate as inactivated vaccine for animals first Agent, there is provided an initiative new application.
The following technical scheme of the present invention is as follows：
A kind of injection-type inactivated vaccine adjuvant for animals, the vaccine adjuvant are 2-8 by mass ratio:98-92 sodium alginate Formed with sterilized water.
Further, described vaccine adjuvant is 2-6 by mass ratio:98-94 sodium alginate and sterilized water composition；
Most preferably, vaccine adjuvant is 4 by mass ratio:96 sodium alginate and sterilized water composition；
Above-mentioned mass ratio why is selected, is due to that inventor is had found when the mixture of sodium alginate and water is as vaccine Adjuvant in use, in order to meet the application requirement of adjuvant, it is necessary to viscosity and toxicity to mixture etc. is reasonably controlled, and It is the requirement for best suiting vaccine adjuvant the characteristics of mixture under above-mentioned mass ratio, meanwhile, alginic acid of the present invention Sodium adjuvant has significant immunoenhancement result compared with white-oil adjuvant, can effectively improve the antibody level, thin of vaccine antigen The humoral immunity such as intracellular cytokine secretion level and lymphopoiesis ability and cellular immune level.
The preferred mean molecule quantity of sodium alginate of the present invention is 1.0 × 105-3.0×105Between sodium alginate.
In addition, inventor additionally provide the adjuvant preparation method it is as follows：
After weighing 100 DEG C of -120 DEG C of hot air sterilization 20min of sodium alginate solid powder, it is well mixed with sterilized water, uses machine Tool method stirs, and sodium alginate is completely dissolved into transparent viscous liquid, regulation pH value to 7.0, obtains immunologic adjuvant, 4 DEG C Save backup；
The viscosity of sodium alginate is influenceed by several factors such as molecular weight, temperature, mass fraction, shear rates.And The sodium alginate adjuvant of above-mentioned preparation initial viscosity when high concentration (4wt%) is prepared is larger, but because it belongs to water-soluble thing Matter, viscosity can substantially reduce after being mixed with antigen, after being configured to vaccine, its kinematic viscosity<30m2/s；And conventional vaccine is used White oil is about 7-10m in 50 DEG C of kinematic viscosity2/ s, and the surfaces such as Span-80 and aluminum stearate must be also added when in use To maintain the stability after adjuvant and antigen liquid emulsification, at this moment its viscosity can be multiplied activating agent, and be needed after being mixed with antigen Emulsified, the viscosity after emulsification can also increase into several times, when its final viscosity is far above above-mentioned use sodium alginate adjuvant 's<30m2/ s, so that the vaccine made is sticky, injection difficulty increase；Therefore although adjuvant provided by the present invention is initially viscous Degree increased compared with white oil, but the vaccine viscosity finally obtained in specific application is far below existing white-oil adjuvant on the contrary, Injection is more suitable for use.
Wherein described machinery emulsification method uses equipment to be completed for colloid mill, homogenizer or refiner, with the spy of mixture Property, which reaches above-mentioned standard, to be advisable；
The present invention still further provides the application method of the vaccine adjuvant, be gone out with H9N2 subtype avian influenza virus injection-types Exemplified by live vaccine, comprise the following steps that：
The surfactant that mass ratio is 4% is added into the H9N2 subtype avian influenza virus chick embryo allantoic liquids of harvest to tell Temperature -80, fully mix, then with the formalin-inactivated 48h of final concentration 0.2%, obtain H9N2 subtype avian influenza virus antigen liquids, to Immunologic adjuvant is added in antigen liquid, is sufficiently mixed uniformly with conventional method, the immunologic adjuvant is with antigen aqueous solution volume ratio 1:1；
The above method is one kind in numerous application methods, can be according to the characteristic of antigen to above-mentioned side during for not synantigen Method carries out accommodation, and inventor will not be repeated here.
Compared with prior art, the present invention achieves following technique effect：
1. live vaccine adjuvant provided by the invention, without toxicity, efficiently solves asking for vaccine side reaction to animal Topic, and easily storage, are easy to inject, easy to use, action effect is good.
2. live vaccine adjuvant provided by the invention, preparation technology is simple, equipment requirement is low, and cost is than traditional white-oil adjuvant More than 5 times are reduced, exemplified by preparing 1L adjuvant costs, 4wt% sodium alginate adjuvant cost about 2-4 members, and white-oil adjuvant (contains Surfactant) cost is more than 15 yuan.Further, since antigen liquid highest content is 50% (i.e. adjuvant in white-oil adjuvant vaccine It is 1 with antigen liquid proportional:1), therefore in order to reach Effective Antigens content, antigen usually requires to concentrate, will so as to improve technology Summation production cost, and live vaccine adjuvant provided by the invention can adjust adjuvant and antigen by changing sodium alginate concentration The proportional quantity of liquid, so as to effectively reduce antigen concentrating degree, further reduce production cost；Live vaccine assistant provided by the invention The stable dosage forms that agent obtains, be not in lamination, and white-oil adjuvant inactivated vaccine using it is preceding needs shake up, environment temperature compared with Pre-temperature is carried out when low, is easily layered when temperature is too high.
3. live vaccine adjuvant provided by the invention, not only there is similar white-oil adjuvant sustained release antigen, also have Significantly increase the function of humoral immunity of organism and cellular immunity.
In summary, live vaccine adjuvant component provided by the invention is simple, easily stores, and modest viscosity, is easy to inject, makes With conveniently, action effect is good, and no side reaction, the developing and utilizingpotentiality in livestock and poultry breeding industry is huge, before having wide application Scape.
Brief description of the drawings
Fig. 1 is that immune effect compares figure in embodiment 4；
Peripheral blood IL-4 concentration compares figure in Fig. 2 embodiments 4；
Peripheral blood IFN-γ concentration compares figure in Fig. 3 embodiments 4；
Peripheral lymphocyte proliferation rate compares figure in Fig. 4 embodiments 4；
Peripheral blood blood lymphocyte ratio compares figure in Fig. 5 embodiments 4.
Embodiment
Presently preferred embodiments of the present invention is described in detail below so that advantages and features of the invention can be easier to by It will be appreciated by those skilled in the art that apparent clearly defined so as to be made to protection scope of the present invention：
It is prepared by the vaccine adjuvant of embodiment 1
Sodium alginate, purchased from Shandong West Asia chemistry limited company, it is faint yellow filament particle or pulverulent solids, divides Minor C5H7O4COONa, molecular weight 1.0 × 105-3.0×105, purity 99%.
It is prepared by sodium alginate adjuvant：Sodium alginate solid powder is weighed, after 100 DEG C of -120 DEG C of hot air sterilization 20min, with nothing The bacterium aqueous solution is well mixed, and is stirred with Mechanical Method, sodium alginate is completely dissolved into transparent viscous liquid, and regulation pH value arrives 7.0, immunologic adjuvant is obtained, 4 DEG C save backup.The mass ratio of sodium alginate and sterilized water is 4:96.
It is prepared by the vaccine of embodiment 2
It is prepared by H9N2 subtype avian influenza virus antigen liquid：9 age in days SPF egg inoculation H9N2 subtype avian influenza virus kinds poison, After 72h, chick embryo allantoic liquid is harvested, virus titer is determined with hemagglutination test, virus liquid is diluted with water to potency as 29, add matter Amount is than being 4:96 Tween-80 is well mixed, and is then added the formaldehyde that mass ratio is 0.2%, is inactivated 48h, as antigen liquid.
It is prepared by vaccine 1-sodium alginate adjuvant H9N2 subtype avian influenzas vaccine：Add in embodiment 1 and obtain into antigen liquid Sodium alginate adjuvant, be sufficiently mixed uniform 20min, the immunologic adjuvant under the conditions of 3000rpm with homogenizer under normal temperature It is 1 with antigen aqueous solution volume ratio:1；
It is prepared by vaccine 2-white-oil adjuvant H9N2 subtype avian influenzas vaccine：Antigen liquid, Bian Jia are slowly added into white-oil adjuvant Side is stirred, and is sufficiently mixed emulsification 30min, the immunologic adjuvant and antigen water under the conditions of 6000rpm with homogenizer under normal temperature Liquor capacity ratio is 1:1.
The security of embodiment 3 compares
From 14 age in days health commercial broilers 180,18 groups, every group 10 are divided into.Vaccination ways, which are divided under wing, to be connect Kind, chest muscle injection and neck are subcutaneously injected, and dosage of inoculation is divided into 1ml, 2ml and 3ml, and different vaccination ways and dosage of inoculation are by just Experiment is handed over to be combined inoculation.The stress reaction of chicken is observed after inoculation in 7 days, and records clinical symptoms and morbidity number of elements.Knot Fruit is shown in Table 1, no matter not causing chicken adverse reaction by which kind of visible vaccination ways of table and dosage of inoculation vaccine 1；Under the wing of vaccine 2 When occurring the symptom of lassitude, chest muscle injection and neck inoculated with subcutaneous injections dosage 1ml during dosage of inoculation 2ml and 3ml A small number of chicken adverse reactions can be caused, neck is subcutaneously injected stress be larger.The above results show, sodium alginate Adjuvanted vaccines compared with High dose will not also cause obvious stress reaction when being inoculated with, and part chicken occurs significantly after white-oil adjuvant vaccine inoculation Stress reaction, when particularly dosage of inoculation is larger.
The security of the vaccine different vaccination ways of table 1 and dosage of inoculation compares
The immune effect of embodiment 4 compares
1 material and method
1.1 experimental animal
75 blue brown chick in health sea are purchased from Taian Shandong chicken house.
1.2 reagents and instrument
Hyclone, DMEM and RPMI1640 cell culture fluids produce for Gibco companies；Dimethyl sulfoxide (DMSO) (DMSO), thiophene Azoles indigo plant (MTT), chicken lymphocyte separation medium, cleaning fluid, concanavaline (ConA) produce for Sigma companies；CD4+ and CD8+ antibody dyestuffs, the production of Biolegend companies of the U.S.；IFN-γ, IL-4ELISA detection kits Shanghai Lang Dun biotech firms Production.
1.3 experimental designs and Testing index
Chicken is randomly divided into 3 groups, every group 25.
Experiment packet：
I groups：Use the live vaccine 1 of the embodiment of the present invention 2.
II groups：Use the live vaccine 2 of the embodiment of the present invention 2.
III groups：Without Adjuvanted vaccines control group.
Vaccine inoculation：Each group chicken vaccine injection dosage is every 1ml, respectively 7 after immune, 14,21,28,35,42,49d Blood sampling, suppress (HI) testing inspection serum antibody titer with blood clotting (HA) and blood clotting, with ELISA method detection Cytokine of Serum Level, with Automatic Blood Cell Analyzer determine PBLC ratio, with PI decoration methods detect blood lymphocytes Proliferation rate.All data carry out Duncan ' s Multiple range tests with the softwares of SPSS 17.0 and analyzed, and data are average value ± SD, P< 0.05 represents significant difference.
2 results and analysis
2.1 serum antibody titers compare
Each test group is 7 after immune, 14,21,28,35,42,49d randomly select 3 chickens, venous blood collection 1mL under wing, point From serum, antibody titer is detected with blood clotting and hemagglutination-inhibition test, is as a result represented with log2.As a result Fig. 1, vaccine I group antibody are seen 21d peaks, and vaccine II groups and control group 28d peak；High titre (the P of vaccine I group antibody peakedness ratio vaccine II groups <0.05), this gap can maintain 49d (P<0.05).As a result show, the vaccine-induced body antibody level of white-oil adjuvant is notable Higher than the control group for not adding adjuvant, and the antibody level of sodium alginate Adjuvanted vaccines induction is integrally higher than white-oil adjuvant vaccine, carries Preceding antibody peak value node, and antibody titers can be maintained for a long time.Therefore, sodium alginate adjuvant, which has, improves humoral immunity level Function.
2.2 cytokines concentration compare
Every group randomly selects 3 chicken blood samplings in above-mentioned time point, and it is standby to centrifuge serum.Using chicken IL-2 gene, IL-4 and The content of each cell factor in the ELISA kit measure serum of IFN-γ, OD450nm absorbance is surveyed with ELIASA, according to The concentration of standard items and corresponding OD values calculate the linear regression equation of standard curve, according still further to sample OD values in recurrence side Corresponding sample concentration is calculated in journey.Specific method is carried out according to kit specification.As a result Fig. 2 and Fig. 3 are seen, vaccine I groups, II groups and III group peripheral blood IL-4 concentration reach peak value in 21d；Vaccine I groups and II group IL-4 concentration are notable since 7d Higher than III groups (P<0.05), and vaccine I group integral levels are significantly higher than vaccine II groups (P since 21d<0.05).Vaccine I groups, II groups and III group peripheral blood IFN-γ concentration reach peak value in 28d；Vaccine I group numerical value is all remarkably higher than II groups since 14d (P<0.05), II groups are significantly higher than III groups (P<0.05).As a result show, sodium alginate Adjuvanted vaccines and white-oil adjuvant vaccine lure Lead body cell factor level and be significantly higher than the control group for not adding adjuvant, and the antibody level of sodium alginate Adjuvanted vaccines induction is whole Body is higher than white-oil adjuvant vaccine.Sodium alginate adjuvant has the function of promoting cytokine secretion.
2.3 peripheral lymphocyte proliferation rates compare
Isolated lymphocyte as described above, 1 × 10 is adjusted to by its density6/ mL, 96 holes are inoculated in 100 μ L/ holes Culture plate, with the RPMI-1640 medium cultures containing 10% hyclone.3 holes add 25uL ConA (20mg/mL) before each column Solution, 3 holes are set to control wells and add 25uL PBS, 5%CO after each column2, be incubated after 24h under the conditions of 37 DEG C and add 10 μ L MTT per hole, 4h is incubated, discards nutrient solution, 100mL DMSO are added per hole, ELIASA surveys OD490nm absorbance.Lymphocyte transformation rate (LTR) calculated with following equation：LTR=(ConA stimulations average-without ConA stimulations average)/without ConA stimulation averages.As a result see Fig. 4, vaccine I groups and II group lymphocytic proliferation rates are all remarkably higher than III groups (P since 7d<0.05) epidemic disease, and since 14d Seedling I group integral levels are significantly higher than vaccine II groups (P<0.05).As a result show, sodium alginate adjuvant, which has, promotes lymphocyte to increase The function of growing.
2.4 PBLC ratios compare
Every group randomly selects 3 chickens in above-mentioned time point, gathers 1mL anticoagulations respectively, separates lymphocyte, uses cleaning fluid Wash 2 times, its density is adjusted to 1 × 106/mL.50uL lymphocyte liquid is taken, adds each 10uL of CD4+, CD8+ dyestuff, 4 DEG C are kept away Light action 20min.Using the percentage of flow cytometer measure CD4+, CD8+ cell, CD4+/CD8+ ratios are calculated.As a result see Fig. 5, vaccine I groups and II group CD4+/CD8+ ratios are all remarkably higher than III groups (P since 14d<0.05) epidemic disease, and since 14d Seedling I group integral levels are significantly higher than vaccine II groups (P<0.05).As a result show, sodium alginate adjuvant, which has, promotes T lymphocytes The immunologic function of mediation.
Embodiment 5 is attacked malicious protecting effect and compared
30 blue brown chick in health sea, are equally divided into 3 groups.Above-mentioned three kinds of vaccines are immunized respectively at 14 ages in days, after immune 42d, to each group chicken with 106TCID50The H9N2 viruses of dosage carry out intravenous injection and attack poison, observe and record the clinical condition for attacking malicious chicken Shape.The clinical symptoms of reference include lassitude, loss of appetite, nasal mucus, shed tears, swollen head, expiratory dyspnea, rale, feather are fluffy and disorderly, Diarrhea, death etc..2 are the results are shown in Table, vaccine I groups 10 attack malicious chicken without clinical symptoms；Vaccine II groups 10 attack malicious 2 appearance of chicken The symptoms such as lassitude, loss of appetite, feather be fluffy and disorderly；Vaccine III groups 10 attack malicious chicken 4 only occur lassitude, loss of appetite, The symptoms such as feather is fluffy and disorderly.As a result show, sodium alginate Adjuvanted vaccines have more preferable immune protective effect than white-oil adjuvant vaccine.
The vaccine protest test of table 2
It is prepared by the different proportion vaccine adjuvant of embodiment 6
The method recorded according to embodiment 1 prepares sodium alginate adjuvant；The mass ratio of sodium alginate and sterilized water is 2:98 As adjuvant I；The mass ratio of sodium alginate and sterilized water is 8:92 as adjuvant II；
Above-mentioned identical checking test is carried out by adjuvant is obtained in the adjuvant of above-mentioned acquisition and embodiment 1, as a result finds it Although effect has a certain distance compared with the adjuvant in embodiment 1, it is better than the experimental group using white-oil adjuvant, it is seen that this hair Bright provided adjuvant proportioning effect is better than white-oil adjuvant.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Claims (5)

  1. A kind of 1. injection-type inactivated vaccine adjuvant for animals, it is characterised in that：The vaccine adjuvant is 2-8 by mass ratio:98-92's Sodium alginate and sterilized water composition.
  2. 2. injection-type inactivated vaccine adjuvant for animals according to claim 1, it is characterised in that described vaccine adjuvant is by matter It is 2-6 to measure ratio:98-94 sodium alginate and sterilized water composition.
  3. 3. injection-type inactivated vaccine adjuvant for animals according to claim 1, it is characterised in that described vaccine adjuvant is by matter Amount is than being 4:96 sodium alginate and sterilized water composition.
  4. 4. the injection-type inactivated vaccine adjuvant preparation method for animals described in claim 1, is comprised the following steps that：
    After weighing 100 DEG C of -120 DEG C of hot air sterilization 20min of sodium alginate solid powder, it is well mixed with sterilized water, uses Mechanical Method Stir, sodium alginate is completely dissolved into transparent viscous liquid, regulation pH value to 7.0, obtain immunologic adjuvant, 4 DEG C of preservations It is standby.
  5. 5. injection-type inactivated vaccine adjuvant preparation method for animals according to claim 4, it is characterised in that described machinery Emulsion process is completed using colloid mill or homogenizer or refiner.
CN201711345593.5A 2017-12-15 2017-12-15 A kind of injection-type inactivated vaccine adjuvant for animals Pending CN107802832A (en)

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Citations (2)

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CN104208029A (en) * 2013-05-30 2014-12-17 上海医药工业研究院 Vaccine composition powder preparation used for nose and preparation method thereof
CN104013955A (en) * 2014-06-18 2014-09-03 中国科学院过程工程研究所 Oil-in-water emulsion free of surfactant and use thereof

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