CN104906574A - Application of chitosan oligosaccharide in preparing vaccine adjuvant and vaccine composition - Google Patents
Application of chitosan oligosaccharide in preparing vaccine adjuvant and vaccine composition Download PDFInfo
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Abstract
The invention discloses an application of chitosan oligosaccharide in preparing a vaccine adjuvant as well as a vaccine composition and a vaccine preparation containing the vaccine adjuvant. The vaccine adjuvant comprises chitosan oligosaccharide. The method for preparing the chitosan oligosaccharide of which the polymerization degree is 2-20 comprises the following steps: performing biological enzymatic hydrolysis, chemical decomposition or physical method decomposition on chitosan, wherein the deacetylation degree of the chitosan oligosaccharide of which the polymerization degree is 2-20 is greater than 90%, the molecular weight is 400-5000Da, and the sum of components of which the polymerization degrees are 3-10 is not less than 50%. The vaccine adjuvant containing chitosan can be used as an adjuvant of an attenuated vaccine, a nucleic acid vaccine, a polypeptide vaccine or protein vaccine.
Description
Technical field
The invention belongs to medical art, particularly, the present invention relates to oligochitosan and preparing vaccine adjuvant and containing the purposes in the vaccine combination of this vaccine adjuvant and bacterin preparation.
Background technology
Adjuvant be a class prior to antigen or apply with antigen simultaneously time can the material of enhancement antigen immunological effect, its effect specific immune response that mainly enhancing human body immunity system counter is former reaction.Before more than 200 years, British physician Jenner inoculates cowpox prevention variola and achieves human trial success, and the mankind can pass through some infectious disease of vaccination prevention and control.Nineteen twenty-five France immunologist hold concurrently veterinary scholar Gaston Ramon find to add in diphtheria, tetanus vaccine material that some and the antigen such as tapioca, agar, suppurative bacterium has nothing to do can specifically enhancing body to the resistance of diphtheria and tetanus toxin.In the research of vaccine adjuvant after this, find that many materials all have adjuvant effect, as tapioca starch, lanoline, tannic acid etc.In recent years, that studies immunological adjuvant along with people deepens continuously, increasing material is found to have the function of immunological adjuvant, as (Biomolecular Engineering such as Alum adjuvant, oil emulsion adjuvant, propolis adjuvant, polysaccharide adjuvant, immunologic adjuvants, 2001,18 (3): 69-85.).Oil emulsion adjuvant mainly contains the Freund adjuvant (FA) of water in oil emulsion, the MF-59 etc. of oil in water emulsion.Immunologic adjuvant has liposome, Cytokine adjuvant etc.So far the Human vaccine adjuvant being ratified legal use by China is Alum adjuvant, and it can induction of immunity reaction effectively, the humoral immunoresponse(HI) (Arch Virol, 2008,153 (5): 831-837.) of remarkable enhancing body.But aluminium adjuvant can only the humoral immune function of enhancing body, effective cellular immunization facilitation cannot be produced for such as born of the same parents' inner virus such as HIV.Secondly, aluminium adjuvant mainly with aluminium hydroxide and aluminum phosphate for representative, produce the local responses such as redness, tuberosity in injection site, aluminum salt is also easily accumulated in vivo, particularly brain, likely causes the generation of encephalopathy.In tradition live vaccine, conventional adjuvant has Alum adjuvant and oily adjuvant.Oil-adjuvant vaccine is all better than Alum adjuvant vaccine in antibody titer and immune persistence, but oily adjuvant untoward reaction is serious, and subcutaneous injection can cause inflammation, ulcer and granuloma; The long-term retention of oily substance not easily metabolism in tissue.
Along with deepening continuously of immunological investigation and developing rapidly of technique for gene engineering, the development of various novel gene engineered vaccine has been carried out for various disease, new technique vaccine is as epitope vaccine, the antigen purity such as recombinant vaccine are more and more higher, but these vaccine ubiquity molecules are little, immunogenicity is weak, be difficult to induction body and produce the deficiencies such as effective immunne response, thus need immunological adjuvant to carry out collaborative this effect, especially safety non-toxic, the adjuvant of stronger cellullar immunologic response can be stimulated, and applicable mucosal vaccine, the immunological adjuvant of nucleic acid vaccine and tumor vaccine.Desirable vaccine adjuvant should have following characteristics: for specific crowd or animal, ensure that side effect is minimum; Adjuvant effect wants lasting stability; Production cost is as far as possible low; The intensity strengthening cell or humoral immunization is wanted to reach protection requirement; The toxic and side effects that different route of administration produces all should know research.
Oligochitosan molecular weight is little, water solublity is good, easily be absorbed by the body, biological activity is high, the unique Physiology and biochemistry had not available for macromole sugar is active, there is pure natural simultaneously, radiationless, the feature such as pollution-free, at health product, nutrient, the food industry aspects such as food additive, the farming and animal husbandry such as plant growth regulator and feed additive aspect, wastewater treatment, weaving, chemical industry, household chemicals, the aspects such as biological engineering and medicine have good using value, more it is worth noting that it can effectively activate Cellular Immunity and humoral immunization, be developed as vaccine adjuvant to have a good application prospect.
The preparation of oligochitosan mainly contains chemical method, physical degradation methods, enzyme process, glycosyl transfer method (Chinese biochemical drug magazine, 1999,20 (2): 99).Chemical method generally uses concentrated hydrochloric acid hydrolysis, but its product is many below tetrose, and chemical reaction condition is very harsh, wayward, and post processing is also loaded down with trivial details.Glycosyl transfer method utilizes low polymerization degree oligosaccharide, after the participation of enzyme (mainly lysozyme or chitinase), extends the oligosaccharide that sugar chain becomes high polymerization degree, and the method mainly obtains six sugar and seven sugar.Enzymatic degradation method enzyme used has chitosanase, cellulase, glycosidase and lipase etc., uncontrollable to its degree of polymerization.And the oligochitosan of different polymerization degree has impact to its adjuvanticity.The current very limited oligosaccharide that can prepare is expensive, and it is very restricted in application.
Summary of the invention
The object of the invention is to, provide oligochitosan preparing vaccine adjuvant and containing the purposes in the vaccine combination of this vaccine adjuvant and bacterin preparation.
For achieving the above object, present invention employs following technical scheme:
A first aspect of the present invention relates to a kind of oligochitosan, and described oligochitosan is prepared by following methods:
It is the oligochitosan of 2-20 that chitosan generates the degree of polymerization through biological enzyme hydrolysis, chemical method degraded or physical method (microwave, roentgenization) cracking;
The described degree of polymerization is the oligochitosan deacetylation >90% of 2-20, and molecular weight ranges is 400 ~ 5000Da, and wherein the degree of polymerization is that the component ratio sum of 3-10 is not less than 20% ~ 50%.
Oligochitosan according to any one of the present invention, it has feature shown in Fig. 1 or Fig. 2.
Another aspect of the invention relates to a kind of vaccine adjuvant, and it comprises above-mentioned oligochitosan; Particularly, described vaccine adjuvant is the adjuvant of attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine.
Another aspect of the invention relates to a kind of bacterin preparation or vaccine combination, and it comprises oligochitosan component of the present invention.
Bacterin preparation according to any one of the present invention or vaccine combination, it is attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine.
Bacterin preparation according to any one of the present invention or vaccine combination, it is hepatitis B subunit vaccine.
Another aspect of the invention relates to oligochitosan described in any one of the present invention and is preparing the purposes in vaccine adjuvant; Or preparing the purposes in vaccine combination or bacterin preparation.
According to the purposes of any one of the present invention, wherein, described vaccine adjuvant is the adjuvant of attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine; Described vaccine is attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine.
The vaccine combination that the present invention relates to, this vaccine combination is containing vaccine antigen and oligochitosan adjuvant;
The bacterin preparation that the present invention relates to, this bacterin preparation is containing the various adjuvants needed for vaccine antigen, oligochitosan adjuvant and preparation;
The invention still further relates to a kind of immunization method or inoculation method, comprise and give the vaccine combination of mammal effective dose or the step of bacterin preparation.
Term " effective dose " refers in mammal and realizes the bacterin preparation of immune effect or the dosage of vaccine combination.
Beneficial effect of the present invention:
Oligochitosan of the present invention can strengthen immunne response, has good adjuvant effect, can be used as the adjuvant of attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine.Oligochitosan in the present invention can large-scale production.The present invention is that the research of oligochitosan adjuvant effect mechanism lays the foundation, and provides reference for finding the oligosaccharide with better adjuvant effect.
The present invention is by biological enzyme hydrolysis, chemical method degraded or physical method cracking, and binding film isolation technics, the preparation degree of polymerization is the oligochitosan of 2-20, can high yield produce on a large scale, and it is quality controllable, cost-effective, for making under internal milieu easily molten, easy absorption, oligochitosan easy to use, can be applied to vaccine adjuvant and providing material base.
Accompanying drawing explanation
Fig. 1 is the Mass Spectrometer Method collection of illustrative plates of oligochitosan of the present invention;
Fig. 2 is that the oligochitosan degree of polymerization of the present invention detects collection of illustrative plates;
Fig. 3 is H1N1 virus antigen compatibility oligochitosan the 1st immune serum antibody titer;
Fig. 4 is H1N1 virus antigen compatibility oligochitosan the 2nd immune serum antibody titer;
Fig. 5 is hepatitis B subunit vaccine compatibility oligochitosan the 1st immune serum antibody titer;
Fig. 6 is hepatitis B subunit vaccine compatibility oligochitosan the 2nd immune serum antibody titer;
Fig. 7 is hepatitis B subunit vaccine compatibility oligochitosan the 2nd immune serum antibody subtype and subclass.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
embodiment 1: the preparation of oligochitosan and physicochemical property
1, oligochitosan preparation:
Add deionized water in material-compound tank, chitosan is dissolved in the acetum of 1%, stir and be warming up to 38 ± 2.0 DEG C.Add the enzyme preparation of 5% (to substrate), stir, 38 DEG C of constant temperature, after enzymolysis solution viscosity is down to 60%, start ultrafiltration (molecular weight cut off is 6000).Be less than the component permeate film of molecular weight cut off, the part being greater than molecular weight cut off returns reactor and continues degraded.Ultrafiltration permeate is again by nanometer film filter (molecular weight cut off 300), permeation parts is that (this permeation parts can be recycled for water, acetic acid and small molecular sugar, for preparing chitosan solution), non-permeation parts is concentrated as oligochitosan solution, spraying dry, obtained oligochitosan powder.
2, oligochitosan Mass Spectrometer Method:
Instrument: BIFLEX III type MALDI-TOF mass spectrograph (Bruker company);
Method: oligochitosan sample is made into the solution of 10mg/mL, substrate uses 2,5-resorcylic acid (DHB), draw 1uL matrix solution and sample solution respectively, abundant mixing, draws the mixing drop of 0.5uL in clean rustless steel sample panel, after the crystallization of solvent nature volatile samples, carry out mass spectral analysis, result as shown in Figure 1.As seen from the figure: the COS degree of polymerization is 2-20.
3, the oligochitosan degree of polymerization detects:
Sample treatment: 8% (W/V) oligochitosan by the degree of polymerization being 2-8 is water-soluble, regulates pH>12 with ammonia, cross elimination precipitation, supernatant is analyzed.
Testing conditions: HPLC ELSD detector and Clici Mal chromatographic column, linear gradient type of elution; Column temperature 30 DEG C; Detector air pressure 23psi; Flow velocity: 1ml/min.
Mobile phase: A, ammonium formate solution; B, acetonitrile.
Elution program: 0-30 minute, 70%B to 50%B.
Experimental result: adopt area normalization method to calculate different polymerization degree chitooligose monomer proportion in this sample and be followed successively by 17.8%, 27.2%, 26.8%, 16.7%, 7.0%, 2.7% and 1.9%, as shown in Figure 2.
embodiment 2: oligochitosan measures the adjuvanticity of influenza vaccines
Using the oligochitosan in embodiment 1 as vaccine adjuvant, be antigen with H1N1 influenza vaccines (H1N1 influenza virus cracking liquid, 30 μ g/ml), the two coupling intramuscular injection immune mouse, measures antibody titer.Concrete grammar is as follows:
Laboratory animal: BALb/c mice, 6-8 week age, 5/group, female.
Drug level: the oligochitosan (COS) by preparation in above-described embodiment 1: 4mg/ml and 20mg/ml; H1N1 influenza virus cracking liquid: 30 μ g/ml; Aluminium adjuvant (Al): 4mg/ml.
Control solvent: normal saline (Saline).
Drug level after mixing: oligochitosan: 2mg/ml and 10mg/ml; H1N1 influenza virus cracking liquid: 15 μ g/ml; Aluminium adjuvant (Al): 2mg/ml.
Dosage: oligochitosan: 200 μ g/mouse (mice) and 1mg/mouse; H1N1:1.5 μ g/mouse; Aluminium adjuvant (Al): 200 μ g/mouse.
Grouping: (1) blank group: Saline; (2) P group: Saline+H1N1; (3) aluminium adjuvant group: Al+H1N1; (4) COS low dose group: COS (200 μ g/mouse)+H1N1; (5) COS high dose group: COS (1mg/mouse)+H1N1.
Equal-volume mixing before injection, 100 μ l/ Mus, right hind intramuscular injection.
Immunization protocol: animal was according to latter 2 weeks of immunity grouping first immunisation injection, and tail venous blood sampling, measures Serum Antibody titre.4th week booster immunization after first immunisation, after secondary immunity 2 weeks, tail venous blood sampling, measured Serum Antibody titre.Depending on titre situation, after measuring, within 2 weeks, carry out the 3rd immunity.ELASI method measures Serum Antibody titre.
ELISA method agents useful for same is prepared:
1. antigen coated liquid: 50mmol/L carbonate buffer solution, pH9.6.Take anhydrous Na2CO3 1.696g, NaHCO3 2.856g, be dissolved in water 1000mL, PH 9.6.
2. cleaning mixture (10 × PBST, PH 7.4): take NaCl 80g, KCl 2g, Na
2hPO4 29g, KH
2pO4 2g, Tween-20 10mL, adds distilled water to 1000ml, is adjusted to PH 7.4, dilutes 10 times during use.
3. confining liquid: 1%BSA, dissolves with 50mmol/L PBS PH 7.4.
4. substrate solution A (TMB-hydrogenperoxide steam generator): get 3,3`, 5,5`-tetramethyl benzidine (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL.
5. substrate solution B (0.1mol/L citric acid-0.2mol/LNa2HPO4 buffer): Na2HPO4 24.8g, citric acid 19.33g, add distilled water to 1000ML, regulates pH to 5.0 ~ 5.4.
6. substrate solution: during use, substrate solution A and substrate solution B is pressed 1:1, add 30%H
2o
2to final concentration 0.5%.
7. stop buffer: 2N H
2sO4.
The antigen coated liquid of H1N1 antigen is diluted to 4 μ g/ml, adds in 96 orifice plates (Costar), 100 μ l/ holes, and 4 DEG C of bags are spent the night.3 times are washed, 1%BSA, 200 μ l/ holes, 37 DEG C of closed 1h with PBST.Wash 3 times with PBST, mouse resisting anteserum is from 1:400, and with PBST by equimultiple doubling dilution 6 gradients, 100 μ l/ holes add ELISA Plate, hatch 1h for 37 DEG C.Discard blood serum sample, PBST washs 3 times, adds goat anti-mouse IgG-HRP antibody (PBST 1:1000 doubly dilutes), adds in ELISA Plate, and 100 μ l/ holes, hatch 1h for 37 DEG C.Discard two to resist, PBST washs 6 times, adds the fresh tmb substrate nitrite ion of new configuration, 100 μ l/ holes, room temperature lucifuge colour developing 15min.Add 2N sulfuric acid solution color development stopping, 50 μ l/ holes, and measure 450nm absorbance (A450) by microplate reader.
Experimental result:
With H1N1 virus lysate for immunizing antigen, oligochitosan can produce the specific antibody (Fig. 3) of higher titre as adjuvant initial immunity.Through second time immunity, oligochitosan has significant immunolgical adjuvant activity, can the generation (Fig. 4) of enhancing antibody.
embodiment 3: oligochitosan is to the adjuvanticity of Hepatitis B virus vaccine
Using the oligochitosan in embodiment 1 as vaccine adjuvant, be antigen with hepatitis B subunit vaccine of recombinating (expressed by Hansenula yeast HBsAg, Dalian Han Xin biological medicine company limited produces), the two coupling intramuscular injection immune mouse, measures antibody titer.Specific experiment is with embodiment 2.
Dosage: oligochitosan: 200 μ g/mouse (mice) and 50 μ g/mouse; HBsAg:2 μ g/mouse; Aluminium adjuvant (Al): 200 μ g/mouse.
Experiment grouping: (1) normal saline group (Saline); (2) aluminium adjuvant group (Al+HBsAg); (3) hepatitis B subunit group (Saline+HBsAg); (4) COS low dose group: COS (200 μ g/mouse)+HBsAg; (5) COS high dose group: COS (1mg/mouse)+HBsAg.
Equal-volume mixing before injection, 100 μ l/ Mus, right hind intramuscular injection.
Immunization protocol: animal muscle is injected, initial immunity is ELASI method mensuration mice serum antibody titer after 2 weeks, then carries out 2 immunity.2 immunity measure antibody titer and hypotype and subclass again after 2 weeks.
ELISA method detect serum specific antibody hypotype and subclass experimental technique as follows:
The antigen coated liquid of HBsAg antigen is diluted to 4 μ g/ml, adds in 96 orifice plates (Costar), 100 μ l/ holes, and 4 DEG C of bags are spent the night.3 times are washed, 1%BSA, 200 μ l/ holes, 37 DEG C of closed 1h with PBST.Wash 3 times with PBST, after mouse resisting anteserum PBST carries out 1:400 dilution, 100 μ l/ holes add ELISA Plate, hatch 1h for 37 DEG C.Discard blood serum sample, PBST washs 3 times, and after goat anti-mouse Subclass Antibodies PBST1:1000 doubly dilutes, add in 96 hole ELISA Plate, 100 μ l/ holes, hatch 1h for 37 DEG C.PBST washs, and wash 3 times, the rabbit anti goat igg antibody PBST 1:1000 of HRP labelling doubly dilutes, and add in ELISA Plate, 100 μ l/ holes, hatch 1h for 37 DEG C.PBST washs 5 times, adds TMB chromogenic substrate, 100 μ l/ holes, room temperature lucifuge colour developing 15min.Add 2N sulfuric acid solution color development stopping, 50 μ l/ holes, and measure 450nm absorbance (A450) by microplate reader.
Experimental result: with hepatitis B subunit vaccine of recombinating for immunizing antigen, oligochitosan can produce the specific antibody (Fig. 5) of higher Hepatitis B virus vaccine as adjuvant initial immunity.Through second time immunity, the aluminium adjuvant of oligochitosan and Isodose has close immunolgical adjuvant activity, can the generation (Fig. 6) of enhancing antibody, the generation of antagonist IgG hypotype and IgA all has facilitation, promotes (Fig. 7) especially to the generation of IgG1, IgG2a, IgG2b Subclass Antibodies.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (10)
1. a vaccine adjuvant, it comprises oligochitosan, and described oligochitosan is prepared by following methods:
It is the oligochitosan of 2-20 that chitosan generates the degree of polymerization through biological enzyme hydrolysis, chemical method degraded or physical method cracking;
The described degree of polymerization is the oligochitosan deacetylation >90% of 2-20, and molecular weight ranges is 400 ~ 5000Da, and wherein the degree of polymerization is that the component ratio sum of 3-10 is not less than 50%.
2. vaccine adjuvant according to claim 1, is characterized in that, described vaccine adjuvant is the adjuvant of attenuated vaccine, inactivated vaccine, nucleic acid vaccine, polypeptide vaccine or protein vaccine.
3. a vaccine combination, it comprises the vaccine adjuvant described in claim 1.
4. vaccine combination according to claim 3, is characterized in that, described vaccine combination is attenuated vaccine, inactivated vaccine, nucleic acid vaccine, polypeptide vaccine or protein vaccine.
5. vaccine combination according to claim 3, is characterized in that, described vaccine combination is hepatitis B subunit vaccine.
6. the vaccine combination according to any one of claim 3-5, is characterized in that, described vaccine combination is bacterin preparation.
7. an oligochitosan is preparing the purposes in vaccine adjuvant; Or preparing the purposes in vaccine combination and bacterin preparation.
8. purposes according to claim 7, is characterized in that, described vaccine adjuvant is the adjuvant of attenuated vaccine, inactivated vaccine, nucleic acid vaccine, polypeptide vaccine or protein vaccine.
9. purposes according to claim 7, is characterized in that, described vaccine combination is bacterin preparation.
10. the purposes according to claim 7 or 9, is characterized in that, described bacterin preparation is the vaccine of injection in oral, intravenous injection, intra-arterial injection, mucosa delivery, intramuscular injection, subcutaneous injection, organ injection or splanchnocoel.
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| CN105797153A (en) * | 2016-05-02 | 2016-07-27 | 浙江农林大学 | Veterinary vaccine immunologic adjuvant as well as preparation and application method thereof |
| CN106267185A (en) * | 2016-09-19 | 2017-01-04 | 中国科学院过程工程研究所 | The oligochitosan vaccine adjuvant of a kind of chemically based coupling and application thereof |
| CN107648603A (en) * | 2017-09-22 | 2018-02-02 | 中国科学院过程工程研究所 | The purposes of vaccine adjuvant, vaccine combination and sulphation chitosan oligosaccharide in vaccine adjuvant and vaccine combination is prepared |
| CN109248312A (en) * | 2018-10-22 | 2019-01-22 | 四川大学 | Chitosan oligosaccharide is coated with sheep Listeria, preparation method and application |
| CN110613843A (en) * | 2019-10-22 | 2019-12-27 | 武汉轻工大学 | Preparation method of water-soluble chitosan liposome adjuvant for livestock |
| WO2022226672A1 (en) * | 2021-04-30 | 2022-11-03 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method therefor |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105797153A (en) * | 2016-05-02 | 2016-07-27 | 浙江农林大学 | Veterinary vaccine immunologic adjuvant as well as preparation and application method thereof |
| CN106267185A (en) * | 2016-09-19 | 2017-01-04 | 中国科学院过程工程研究所 | The oligochitosan vaccine adjuvant of a kind of chemically based coupling and application thereof |
| CN106267185B (en) * | 2016-09-19 | 2020-01-10 | 中国科学院过程工程研究所 | Chitosan oligosaccharide vaccine adjuvant based on chemical coupling and application thereof |
| CN107648603A (en) * | 2017-09-22 | 2018-02-02 | 中国科学院过程工程研究所 | The purposes of vaccine adjuvant, vaccine combination and sulphation chitosan oligosaccharide in vaccine adjuvant and vaccine combination is prepared |
| CN109248312A (en) * | 2018-10-22 | 2019-01-22 | 四川大学 | Chitosan oligosaccharide is coated with sheep Listeria, preparation method and application |
| CN109248312B (en) * | 2018-10-22 | 2021-03-23 | 四川大学 | Chitosan oligosaccharide coated sheep listeria, preparation method and application |
| CN110613843A (en) * | 2019-10-22 | 2019-12-27 | 武汉轻工大学 | Preparation method of water-soluble chitosan liposome adjuvant for livestock |
| WO2022226672A1 (en) * | 2021-04-30 | 2022-11-03 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method therefor |
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