CN105031644A - Vaccine adjuvant as well as vaccine composition and vaccine preparation both containing vaccine adjuvant - Google Patents
Vaccine adjuvant as well as vaccine composition and vaccine preparation both containing vaccine adjuvant Download PDFInfo
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Abstract
The invention discloses a vaccine adjuvant as well as a vaccine composition and a vaccine preparation both containing the vaccine adjuvant. The vaccine adjuvant comprises chitooligosaccharides which is prepared from the following method: chitosan is subjected to hydrolysis using a biological enzyme method, degradation using a chemical method, or schizolysis using a physical method to produce chitosan oligosaccharide with the polymerization degree of 2-10; alternatively, chitin is directly subjected to degradation using an enzymic method or a chemical method to obtain chitooligosaccharides with the polymerization degree of 2-10; the acetylation degree of chitooligosaccharides is higher than 90%, and the molecular weight range is 400-2000 Da. The vaccine adjuvant provided by the invention can be used as the adjuvant of attenuated vaccine, inactivated vaccine, DNA vaccine, protein vaccine and polypeptide vaccine.
Description
Technical field
The invention belongs to medical art, relate to a kind of vaccine adjuvant and containing 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 GastonRamon 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 (BiomolecularEngineering 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) (ArchVirol, 2008,153 (5): 831-837.) of remarkable enhancing body.But Alum 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, Alum 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.
Chitin oligosaccharide 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 (Du Yuguang, Bai Xuefang, Yu Xingju. the research of oligomerization saccharide substance physiological activity. Chinese biochemical drug magazine, 1997, 18 (5): 268.), 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 chitin oligosaccharide 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 chitinase, chitosanase, cellulase, glycosidase and lipase etc., uncontrollable to its degree of polymerization.And the chitin oligosaccharide 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 a kind of vaccine adjuvant and containing 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 chitin oligosaccharide with following feature:
Chitosan is degraded through biological enzyme hydrolysis or chemical method or physical method (microwave, roentgenization) the cracking generation degree of polymerization is the oligochitosan of 2-10, and reacted by the highly selective acylation of this oligochitosan through amino, the preparation degree of polymerization is the chitin oligosaccharide of 2-10;
Or, utilize enzyme process or the direct degrade chitin of chemical method to obtain the chitin oligosaccharide of the 2-10 degree of polymerization.
The acetyl degree >90% of prepared chitin oligosaccharide, molecular weight ranges is 400 ~ 2000Da.
According to chitin oligosaccharide 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 chitin oligosaccharide above-mentioned in the present invention; 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 vaccine combination or bacterin preparation, and it comprises the vaccine adjuvant containing chitin oligosaccharide component of the present invention.
Vaccine combination according to any one of the present invention or bacterin preparation, it is attenuated vaccine, inactivated vaccine, protein vaccine, nucleic acid vaccine or polypeptide vaccine.
Vaccine combination according to any one of the present invention and bacterin preparation, it is Porcine reproductive and respiratory syndrome (pig blue-ear disease) inactivated vaccine or hepatitis B subunit vaccine.
Another aspect of the invention relates to the above-mentioned chitin oligosaccharide of the present invention and is preparing the purposes in vaccine adjuvant; Or preparing the purposes in vaccine combination and 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 bacterin preparation or vaccine combination are attenuated vaccine, inactivated vaccine, nucleic acid vaccine, protein vaccine or polypeptide vaccine.
The vaccine combination that the present invention relates to, this vaccine combination is containing vaccine antigen and chitin oligosaccharide adjuvant;
The bacterin preparation that the present invention relates to, this bacterin preparation is containing the various adjuvants needed for vaccine antigen, chitin oligosaccharide adjuvant and preparation;
The invention still further relates to a kind of immunization method or inoculation method, comprise and give the bacterin preparation of mammal effective dose or the step of vaccine combination.
Term " effective dose " refers in mammal the dosage of bacterin preparation or the vaccine complex realizing immune effect.
Beneficial effect of the present invention:
Chitin oligosaccharide 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.Chitin oligosaccharide in the present invention can large-scale production.The present invention is that the research of chitin oligosaccharide 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 2-10, is reacted by the highly selective acylation of this oligochitosan through amino, and the preparation degree of polymerization is the chitin oligosaccharide of 2-10.High yield can to produce on a large scale, and quality controllable, cost-effective, for making under internal milieu easily molten, easy absorption, chitin oligosaccharide easy to use, vaccine adjuvant can be applied to and provide material base.
Accompanying drawing explanation
Fig. 1 is that chitin oligosaccharide of the present invention and oligochitosan infrared detection collection of illustrative plates contrast;
Fig. 2 is the mass spectral analysis collection of illustrative plates of chitin oligosaccharide of the present invention;
Fig. 3 is Porcine reproductive and respiratory syndrome killed vaccine antigen compatibility chitin oligosaccharide the 1st immune serum antibody titer;
Fig. 4 is pig blue-ear disease killed vaccine antigen compatibility chitin oligosaccharide the 2nd immune serum antibody titer;
Fig. 5 is pig blue-ear disease killed vaccine antigen compatibility chitin oligosaccharide the 3rd immune serum antibody titer;
Fig. 6 is pig blue-ear disease killed vaccine antigen compatibility chitin oligosaccharide the 3rd immune serum antibody subtype and subclass;
Fig. 7 is hepatitis B subunit vaccine compatibility chitin oligosaccharide the 2nd immune serum antibody titer;
Fig. 8 is hepatitis B subunit vaccine compatibility chitin oligosaccharide 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 chitin oligosaccharide and physicochemical property
1, chitin oligosaccharide 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.
Selective polymerization degree is prepared the chitin oligosaccharide of high acetyl degree at the oligochitosan of 2-10, and oligochitosan is water-soluble, adds excessive acetic anhydride, then adds DMAP catalyst, and 60 DEG C are reacted 4 hours; After having reacted, centrifugal to reactant liquor, get supernatant, then spraying dry, obtain chitin oligosaccharide.
2, chitin oligosaccharide infrared spectrum detects:
By infrared spectrum analysis, as shown in Figure 1, as seen from Figure 1, spectrogram and the chitinous spectrogram of prepared chitin oligosaccharide are basically identical, and the main distinction of the oligochitosan deacetylated with height is 1640cm for the spectrogram of above-mentioned prepared chitin oligosaccharide
-1peak strengthens.
3, chitin oligosaccharide Mass Spectrometer Method:
Instrument: BIFLEX III type MALDI-TOF mass spectrograph (Bruker company);
Method: chitin oligosaccharide sample is made into the solution of 10mg/mL, substrate uses 2,5-resorcylic acid (DHB), draw 1 μ L matrix solution and sample solution respectively, abundant mixing, the mixing drop drawing 0.5 μ L in clean rustless steel sample panel, after the volatile samples crystallization of solvent nature, carry out mass spectral analysis, result is as Fig. 2.
Can draw by Fig. 2: the degree of polymerization of prepared chitin oligosaccharide is 2-10.
embodiment 2: chitin oligosaccharide measures (one) to the adjuvanticity of pig blue-ear disease inactivated vaccine
Using the chitin oligosaccharide in embodiment 1 as vaccine adjuvant, with pig blue-ear disease inactivated vaccine (PRRSV, 1.6mg/ml) for antigen, the two coupling intramuscular injection immune mouse, measures antibody titer.Concrete grammar is as follows:
Laboratory animal: BALB/c mouse, 6-8 week age, 6/group, female, purchased from Beijing dimension tonneau China laboratory animal Technology Co., Ltd., credit number SCXK (capital) 2012-0001.
Drug level: the chitin oligosaccharide (COSNAC) by preparation in above-described embodiment 1: 4mg/ml; Pig blue-ear disease inactivated vaccine (PRRSV): 1.6mg/ml; ISA206 (Seppic company, oily adjuvant)
Control solvent: normal saline (Saline).
Drug level after mixing: chitin oligosaccharide: 2mg/ml; Pig blue-ear disease inactivated vaccine: 800 μ g/ml.
Dosage: chitin oligosaccharide: 200 μ g/mouse (mice); Pig blue-ear disease inactivated vaccine: 80 μ g/mouse; ISA206 (positive control) mixes with pig blue-ear disease inactivated vaccine 1:1 equal-volume, and 31 DEG C are stirred 0.5h.
Grouping: (1) blank group: Saline; (2) P group: Saline+PRRSV; (3) COSNAC group: COSNAC (200 μ g/mouse)+PRRSV; (4) positive controls: ISA206+PRRSV.
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 mouse tail veins get blood, measure 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 Na
2cO
31.696g, NaHCO
32.856g, be dissolved in water 1000mL, PH9.6.
2. cleaning mixture (10 × PBST, PH7.4): take NaCl80g, KCl2g, Na
2hPO429g, KH
2pO42g, Tween-2010mL, add distilled water to 1000ml, be adjusted to PH7.4, dilutes 10 times during use.
3. confining liquid: 1%BSA, dissolves with 50mmol/LPBSPH7.4.
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/LNa
2hPO4 buffer): Na
2hPO424.8g, 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: 2NH
2sO4.
The N protein antigen of PRRSV is diluted to 5 μ g/ml with antigen coated liquid, 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 (PBST1:1000 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 pig blue-ear disease inactivated vaccine for immunizing antigen, chitin oligosaccharide can produce the specific antibody of higher titre as adjuvant initial immunity, as shown in Figure 3.Through second time immunity, chitin oligosaccharide can the generation of enhancing antibody, as shown in Figure 4.Through third time immunity, chitin oligosaccharide has significant immunolgical adjuvant activity, can the generation of enhancing antibody, as shown in Figure 5.
embodiment 3: chitin oligosaccharide measures (two) to the adjuvanticity of pig blue-ear disease inactivated vaccine
Using the chitin oligosaccharide in embodiment 1 as vaccine adjuvant, with pig blue-ear disease inactivated vaccine (PRRSV, 1.6mg/ml) for antigen, the two coupling intramuscular injection immune mouse, measures antibody subtype and subclass.Specific experiment is with embodiment 2.
Animal muscle is injected, and initial immunity carries out 2 immunity for 4 weeks, and 2 times epidemic disease carries out 3 immunity for 4 weeks, and 3 immunity measure mice serum antibody subtype and subclass after 2 weeks again.
ELISA method detect serum specific antibody hypotype and subclass experimental technique as follows:
The N protein antigen of PRRSV is diluted to 5 μ g/ml with antigen coated liquid, 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 PBST1:1000 of HRP labelling doubly dilutes, and adds in ELISA Plate, and 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: through the 3rd immunity, chitin oligosaccharide and ISA206 have close immunolgical adjuvant activity, can the generation of enhancing antibody, as shown in Figure 6, the generation of antagonist IgG hypotype all has facilitation, promotes especially to the generation of IgG1, IgG2a, IgG2b Subclass Antibodies.
embodiment 4: chitin oligosaccharide measures (three) to the adjuvanticity of pig blue-ear disease inactivated vaccine
Using the chitin oligosaccharide in embodiment 1 as vaccine adjuvant, with pig blue-ear disease inactivated vaccine for antigen, the two coupling intramuscular injection immune mouse, measures mouse spleen lymphocyte and subgroup change thereof.
Experiment material: flow cytometry antibody PerCP-CD3e, APC-CD19, PE-CD4, FITC-CD8a, purchased from BD company; Other are with embodiment 2.
Animal muscle is injected, and initial immunity carries out 2 immunity for 4 weeks, and 2 times epidemic disease carries out 3 immunity for 4 weeks, and 3 immunity measure mouse spleen lymphocyte and subgroup change thereof again after 2 weeks.Concrete grammar is as follows:
The each experimental mice of sacrificed by exsanguination, with alcohol-pickled sterilization 3 minutes, spleen is taken out under super-clean bench aseptic condition, be placed on 100 order nylon mesh screens, mesh screen is placed on and fills in the sterilizing glass culture dish of 10mlRPMI1640 culture medium, then with the glass syringe nook closing member grinding spleen of sterilization, make its cell filter mesh screen, make splenocyte suspension.Splenocyte suspension is with 1000r/min, and centrifugal 10min, abandons supernatant, adds 2mlTris-NH
4cl buffer effect 3 ~ 5 minutes splitting erythrocyte, then add the same centrifuge washing of 3ml culture medium 2 times, re-suspended cell again, need more than 95% with Trypan Blue living cell counting.Under biological microscope, carry out cell counting by WBC counting method, each group of splenocyte suspension cell concentration is adjusted to 1 × 10
7/ ml is for subsequent use.Get PBS (washing liquid) 1.0ml that flow cytometer detection pipe 5 respectively adds the 1%FBS that 4 DEG C are deposited, be labeled as 1,2,3,4,5 respectively.Add the Normal group splenocyte 0.1ml that 1 × 107/ml is for subsequent use again, mixing, 1000r/min, 4 DEG C of centrifugal 10min, discard supernatant, add washing liquid 0.1ml to pipe 1, add the APC rat anti-mouse CD19 of PerCP Hamster anti-mouse CD3e, 5mg/L of 2.5mg/L to pipe 2-5 successively
+, 1.25mg/L PE rat anti-mouse CD4
+single labeling antibody solution 0.1ml with the FITC rat anti-mouse CD8a of 5 μ g/ml, mixes gently.Then often group is made even row 3 streaming detector tubes, adds 4 DEG C of washing liquid 1.0ml deposited, then add respective experimental group for subsequent use 1 × 10
7the splenocyte suspension 0.1ml of/ml, mixing, 1000r/min, 4 DEG C of centrifugal 10min, and supernatant discarded, add mixed traget antibody (being made into the mixture containing PerCP-CD3e2.5 μ g, APC-CD195 μ g, PE-CD41.25 μ g, FITC-CD8a5 μ g in every ml soln by washing liquid) solution 0.1ml, mix gently.Under room temperature, lucifuge hatches 25min, and add washing liquid 1.5ml mixing, 1000r/min, 4 DEG C of centrifugal 10min, and supernatant discarded, each pipe adds washing liquid 500 μ l, is fully shaken up by the cell sample of labelling, on FACScaliber stream type cell analyzer, and counting CD3
+and CD19
+the percentage rate of cell and CD3
+in CD4
+and CD8
+the percentage rate (table 1) of cell.
Table 1 is Flow cytometry pig blue-ear disease killed vaccine antigen compatibility COSNAC the 3rd immune mouse spleen lymphocyte and subgroup thereof
* p<0.05 is compared with Normal group (Saline), * compares p<0.01 with Normal group (Saline), and * * * compares p<0.001 with Normal group (Saline); # compares p<0.05 with antigen control group (Salin+PRRSV),
##compare p<0.01 with antigen control group (Salin+PRRSV), ### compares p<0.001 with antigen control group (Salin+PRRSV);
$p<0.05 is compared with positive controls (ISA206),
$ $p<0.01 is compared with positive controls (ISA206),
$ $ $p<0.001 is compared with positive controls (ISA206).
As can be seen from Table 1, COSNAC can significantly improve immune mouse spleen lymphocyte CD4
+t cell and CD8
+the ratio of T cell, significantly improves CD3
+the content of cell and CD3
+/ CD19
+the ratio of cell.
embodiment 5: chitin oligosaccharide is to the adjuvanticity of Hepatitis B virus vaccine
Using the chitin oligosaccharide in embodiment 1 as vaccine adjuvant, with hepatitis B subunit vaccine (the expressed by Hansenula yeast HBsAg that recombinates, Dalian Han Xin biological medicine company limited produces) be antigen, the two coupling intramuscular injection immune mouse, measures antibody titer.Specific experiment is with embodiment 2.
Dosage: chitin oligosaccharide: 200 μ g/mouse (mice); HBsAg:2 μ g/mouse; Aluminium adjuvant (Al): 200 μ g/mouse.
Experiment grouping: (1) normal saline group (Saline); (2) hepatitis B subunit group (Saline+HBsAg); (3) COSNAC group: COSNAC (200 μ g/mouse)+HBsAg; (4) aluminium adjuvant group: Al (200 μ g/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 PBST1:1000 of HRP labelling doubly dilutes, and adds in ELISA Plate, and 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, chitin oligosaccharide is immune through second time as adjuvant, chitin oligosaccharide has higher immunolgical adjuvant activity than the aluminium adjuvant of Isodose, can the generation (Fig. 7) of enhancing antibody, the generation of antagonist IgG hypotype and IgM all has facilitation, promotes (Fig. 8) 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 chitin oligosaccharide, and described chitin oligosaccharide is prepared by following methods:
Chitosan is degraded through biological enzyme hydrolysis or chemical method or the physical method cracking generation degree of polymerization is the oligochitosan of 2-10, and reacted by the highly selective acylation of this oligochitosan through amino, the preparation degree of polymerization is the chitin oligosaccharide of 2-10;
Or, utilize enzyme process or the direct degrade chitin of chemical method to obtain the chitin oligosaccharide that the degree of polymerization is 2-10;
The acetyl degree >90% of described chitin oligosaccharide, molecular weight ranges is 400 ~ 2000Da.
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 Porcine reproductive and respiratory syndrome inactivated vaccine.
6. the vaccine combination according to any one of claim 3-5, it is bacterin preparation.
7. a chitin oligosaccharide is preparing the purposes in vaccine adjuvant; Or preparing the purposes in vaccine combination, described chitin oligosaccharide is prepared by following methods:
Chitosan is degraded through biological enzyme hydrolysis or chemical method or the physical method cracking generation degree of polymerization is 2-10 oligochitosan, and reacted by the highly selective acylation of this oligochitosan through amino, the preparation degree of polymerization is the chitin oligosaccharide of 2-10;
Or, utilize enzyme process or the direct degrade chitin of chemical method to obtain the chitin oligosaccharide of the 2-10 degree of polymerization;
The acetyl degree >90% of described chitin oligosaccharide, molecular weight ranges is 400 ~ 2000Da.
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 the vaccine of injection in oral, intravenous injection, intra-arterial injection, mucosa delivery, intramuscular injection, subcutaneous injection, organ injection or splanchnocoel.
10. the purposes according to claim 7 or 9, is characterized in that, described vaccine combination is bacterin preparation.
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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 |
| CN107200788A (en) * | 2017-05-11 | 2017-09-26 | 暨南大学 | A kind of quaternary phosphonium chitosan and its application as vaccine immunity adjuvant |
| WO2018211113A1 (en) * | 2017-05-19 | 2018-11-22 | Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet | Immunostimulating, highly pure oligosaccharides |
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