CN110004150A - A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity - Google Patents

A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity Download PDF

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CN110004150A
CN110004150A CN201810863771.1A CN201810863771A CN110004150A CN 110004150 A CN110004150 A CN 110004150A CN 201810863771 A CN201810863771 A CN 201810863771A CN 110004150 A CN110004150 A CN 110004150A
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cpg
vaccine
sequence
oligodeoxynucleotide
immune
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CN110004150B (en
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景志忠
房永祥
赵波
贾怀杰
秦娟
陈国华
何小兵
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Lanzhou Veterinary Research Institute of CAAS
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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
    • A61K39/12—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The CpG oligonucleotide sequences and its application that the invention discloses a kind of with immune-enhancing activity.The present invention provides oligodeoxynucleotide, nucleotides sequence is classified as sequence 1, and all nucleotide are thio-modification in the oligodeoxynucleotide, oligodeoxynucleotide shown in sequence 1 provided by the invention, with strong immunological enhancement；Its residence time in vivo is extended, reduces various hydrolases to its hydrolysis；Preparation method is simple, and quality is easily controllable, is easy to large-scale production；Safety is good, and toxic side effect is low.

Description

A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity
Technical field
The invention belongs to vaccine immunity adjuvant technical fields, and in particular to a kind of CpG oligomerization with immune-enhancing activity Nucleotide sequence and its application.
Background technique
During vaccine development, it is recognized that the successful preparation of vaccine, it is necessary to consider the use of adjuvant, it is necessary to grind The compatibility for studying carefully adjuvant Yu vaccine composition makes the two form stable, safety, the vaccine compound with immunogenicity.Therefore, vaccine Adjuvant research be always vaccine research during important link, it as nonspecific immunity strengthening agent to vaccine inoculation after Effective immune response is induced, vital effect is played.Many substances are attempted as vaccine adjuvant, but only aluminium salt Adjuvant, MA59 (oil-in-water emulsion), MPL (glycolipid), virus-like particle (viral like particle, VLP) are immunized and increase Strong reconstruction influenza virus corpusculum (immunopotentiating reconsitituted influenza virosome, IRIV) and cholera enterotoxin (cholera toxin, CT) goes through to be applied in human vaccination.And 206 oil adjuvants (Montanide ISA 206) is the most widely used adjuvant in animal vaccine.Although above-mentioned adjuvant goes through to be applied to In vaccine preparation, but in practical applications, injection site swelling often occur, granuloma, fever, pain, generation allergy occur etc. Side reaction.In addition, some adjuvants are with high costs.Therefore, wide spectrum, the safe and efficient while easy to produce and ideal that uses are developed Vaccine adjuvant is extremely urgent.
Numerous studies confirm that the CpG DNA of artificial synthesized CpG ODN and some bacteriums make with good Immune-enhancing effect With.CpG refers to by cytimidine (cytosine, C) and guanine (guanine, G) through phosphodiester bond (phosphodiester Bonds, p) each two bases at the dinucleotides that is connected to form, CpG dinucleotides and its end 5' and the end 3' constitute CpG motif (CpG motifs).CpG motif is also referred to as immunostimulatory sequence (immunostimulatory sequence, ISS), and CpG ODN refers to the oligodeoxynucleotide of the CpG motif containing non-methylation.CpG dinucleotides in vertebrate gene group The frequency of occurrences it is lower, mostly methylate, and the occurrence rate of CpG dinucleotides is higher in bacterial genomes and mostly non-methyl Change.The immune system of vertebrate will be thin by pattern recognition receptors (pattern recognition receptors, PRRs) This feature in bacterium genome is identified as a kind of dangerous stimulus signal, and then it is anti-to stimulate body to generate immunoprotection It answers.CpG ODN can promote the maturation and activation of Dendritic Cells, macrophage and B cell, raise CD80, CD86, CD40 With the expression of II molecule of MHC-, promote the secretion of the Thl cytokines such as IL-6, IL-12, IFN-γ, induction body generates Thl Type is immunoreacted (phosphodiester CpG oligonucleotides as adjuvants:polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo.Immunology,2002,106 (1):102-112)。
The basic motif of CpG ODN structure is 5 '-X1X2CGYY-3 ', X1Represent purine, X2Purine or thymidine are represented, Y represents pyrimidine.The study found that the immunostimulatory activity of the simple CpG motif containing 6 deoxynucleotides is extremely weak, to make its hair Wave optimal immunostimulating effect, it is necessary to increase the length of CpG, generally 15-30 base.Meanwhile numerous studies also found, The immunostimulatory activity of CpG ODN is related with its sequential structure feature, main including CpG motif quantity in the sequence and position It sets, whether skeleton has carried out thio-modification, whether having palindrome, the end 5' in sequence, whether the free, end 3' is contained Poly G etc..Can there are one or more CpG motifs, the purine and pyrimidine of CpG motif two sides, CpG in one CpG ODN sequence The difference of base between motif makes different CpG ODN performance heterogeneitys, different Immune-enhancing effect and immunoregulation effect is presented. In addition, CpG ODN, there are also species specificity, effect of the same CpG ODN sequence in different plant species is different, have in different plant species The CpG ODN sequence of effect is also different.(CpG motifs in bacterial DNA and their immune Effects.Annual Review of Immunology, 2002,20:709-760).
CpG ODN through endocytosis take in immunocyte after, by cell on inner body/lysosome TLR9 identification and and its In conjunction with causing TLR9 Dimerized, then Dimerized compound recruits 88 (Myeloid of marrow sample differentiation factor downstream Differentiation factor 88, MyD88) and IL-1 receptor-associated kinase (IL-1receptor-associated Kinase 4, IRAK4), make IRAK4 phosphorylation and with tumor necrosis factor receptor-associated factor (TNF-receptor- Associated factor 6, TRAF6) interaction, and then I kappa b kinase is activated, release NF- κ B enters core, activates a series of cores Transcription factor finally promotes associated immune cells to secrete a series of cell factor and chemotactic factor (CF), triggers sterilization machine intracellular System induces inflammatory reaction, removes or eliminate pathogenic microorganism (the signal transduction pathways of invasion body mediated by the interaction of CpG DNA with Toll-like receptor 9.Seminars in immunology,16(1):17-22)。
CpG ODN has shown good application prospect as a kind of novel, safe and efficient immunologic adjuvant.Use CpG For ODN as the adjuvant immunity gorilla of hepatitis B vaccine or monkey, the ability for generating antibody is 15 times of independent vaccine immunity group, table Bright CpG ODN can be used as good hepatitis B vaccine adjuvant.2017.11.9 day, Dynavax Technologies company announces flag Lower HEPLISAV-B drug obtains U.S. FDA approval, which is used as generation The granted vaccine of the first adjuvant containing CpG in boundary (Dynavax Announces FDA approval of HEPLISAV-B (TM) for prevention of Hepatits B in Adults.http://investors.dynavax.com/events- presentations).Adjuvant of the Coley company by CpG ODN 7909 as hepatitis B vaccine, clinical test results show this Group is applied alone much higher than hepatitis B vaccine in its antibody titer of all time points of vaccine after initial immunity.Coley company is by CpG ODN 7909 as commercialization multivalence vaccinum influenzae inactivatum (Fluarix) adjuvant, I clinical trial phase of randomized double-blind the result shows that, CpG ODN is a kind of safe and reliable adjuvant, can reduce the dosage of (Fluarix).CpG ODN is combined with pseudorabies vaccine and is connect When kind piglet, the reaction of its humoral and cellular immune response can be remarkably reinforced.When CpG ODN and Streptococcus suis septicemia inactivated vaccine are combined, The immune response of pig body can be significantly increased.In short, a large amount of research confirms both at home and abroad, CpG ODN is that one kind is novel, efficiently exempts from Good immunologic adjuvant effect is presented in epidemic disease activator.But the performance of CpG ODN immune-stimulating effect and its sequential structure are close Cut phase is closed, and not homotactic its immunostimulating effect of CpG ODN is also different, moreover, CpG ODN is to different kind, thin Born of the same parents' subgroup shows different effect of stimulation, different immune-stimulating effects is presented.Therefore, it is necessary to further study different sequences The CpG ODN of column is to different genera, cell, the action rule of antigen composition and design and improves out efficient, special CpG ODN Sequence lays the foundation for CpG ODN as application of the vaccine adjuvant in vaccine preparation, and also the prevention and control for great epidemic disease provide skill Art guarantee.
Summary of the invention
It is an object of the present invention to provide a kind of oligodeoxynucleotide.
Oligodeoxynucleotide provided by the invention, nucleotides sequence are classified as sequence 1, and institute in the oligodeoxynucleotide Having nucleotide is thio-modification, which contains 2 CpG units, a length of 21nt of chain.
Above-mentioned oligodeoxynucleotide is also what the present invention protected in the application as immunopotentiator or immunologic adjuvant Range.
Application of the above-mentioned oligodeoxynucleotide in the immunogenicity for improving antigen or vaccine is also present invention protection Range.
Above-mentioned oligodeoxynucleotide is also the scope of protection of the invention preparing the application in vaccine.
Another object of the present invention is to provide a kind of vaccine.
Vaccine provided by the invention comprising immunogene and above-mentioned oligodeoxynucleotide.
Among the above, the vaccine is animal vaccine.
Among the above, the vaccine is animal vaccine, and the animal vaccine is aftosa vaccine.
The invention has the following advantages that
1, artificial synthesized CpG ODN (sequence 1) has strong immunological enhancement；
2, artificial synthesized CpG ODN (sequence 1) extends its residence time in vivo, reduces various hydrolases pair Its hydrolysis；
3, artificial synthesized CpG ODN (sequence 1) preparation method is simple, and quality is easily controllable, is easy to large-scale production；
4, artificial synthesized CpG ODN (sequence 1) safety is good, and toxic side effect is low；
For these reasons, inventor by comparison to CpG ODN sequence, optimization, design, filter out one have it is strong The CpG ODN sequence of strong immunopotentiating ability, and its immune enhancing potential is verified by external, experiment in vivo.
Detailed description of the invention
Fig. 1 is the spleen cell using mouse splitting erythrocyte, is sieved using the method for CFSE labelling method detection T cell proliferation Select one of result figure of CpG ODN sequence.
Fig. 2 is the spleen cell using mouse splitting erythrocyte, is sieved using the method for CFSE labelling method detection B cell proliferation Select one of result figure of CpG ODN sequence.
Fig. 3 is a result figure of the spleen cell secrete cytokines that CpG ODN stimulates mouse splitting erythrocyte.
Fig. 4 is different time points mice serum after single immunization after CpG ODN is mixed with aftosa epidemic disease antigen, 206 adjuvants Antibody titer measurement result figure.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Combined with specific embodiments below, the present invention will be described in detail, but the invention is not limited to these to implement Mode, any improvement or replacement on the present embodiment essence spirit, still falls within the claims in the present invention model claimed It encloses.
The screening of the oligodeoxynucleotide molecule of embodiment 1, the sequence units containing CpG
By the screening and verifying to 4 CpG ODN sequence (table 1) structures effects and function, having filtered out one has strongly The CpG ODN sequence C pG-45-mod of immunological enhancement, nucleotides sequence is classified as sequence 1, and all nucleotide are all thio Modification.
CpG ODN nucleotide backbone in table 1 be all nucleotide without thio-modification (title suffix is-non) and All nucleotide whole thio-modifications (title suffix is-mod).CpG-21 is free of CpG unit, chain length 15nt；CpG-45 contains 2 A CpG unit, chain length 21nt.
Table 1
Group Sequence (5 ' -3 ')
CpG-21-non GCTAGAGCTTAGGCT
CpG-21-mod GCTAGAGCTTAGGCT
CpG-45-non TCCATGACGTTCCTGACGTTG
CpG-45-mod TCCATGACGTTCCTGACGTTG (sequence 1)
Specific immune effect detection is as follows:
1, the separation of Mouse spleen cells
(1) break cervical approach or CO2Anaesthesia puts to death C57BL/6 mouse, and the spleen of sterile separating mouse places it in 100 μm Sieve on, side grinding be added 1640 culture medium of RPMI (Gibco, article No. C11875500BT), filter muscle or its hetero-organization Part.
(2) 2000rpm/min, room temperature are centrifuged 5 minutes.Residual liquid is discarded, 2mL erythrocyte cracked liquid is added, and (Tiangeng is raw Change scientific and technological (Beijing) Co., Ltd, article No. RT122-02), resuspension cell, room temperature 5 minutes.
(3) 1640 culture medium of RPMI of 2-3 times of volume is added immediately, is mixed by inversion.2000rpm/min, room temperature centrifugation 5 Minute.Residual liquid is discarded, 1mL DPBS is added.
(4) final concentration of 5uM CFSE (5 (6)-Carboxyfluorescein diacetate N- are added in appropriate cell Succinimidyl ester, CFSE) (carbox fluorescenceindiacetate succinimidyl ester) (Sigma-Aldrich, article No. 21888)。
(5) it is protected from light, 37 DEG C shake 10 minutes.RPMI 1640/10%FBS (the fetal of 1 times of volume is added immediately Bovine serum, FBS) (fetal calf serum) (Gibco, article No. 16000044), it sets 5 minutes on ice.2000rpm/min, room temperature Centrifugation 5 minutes.Suitable 1640/10% fetal calf serum of RPMI is added, washs 3 times.1mL RPMI1640/10% tire ox is added Serum counts.
(6) by 1 × 106A/mL is inoculated in 48 porocyte culture plates, is separately added into final concentration of 1 μ g/mL CpG-21- Non, CpG-21-mod, CpG-45-non, CpG-45-mod, 100ng/mL positive control LPS (lipopolysaccharide, LPS) (lipopolysaccharides) (Sigma-Aldrich, article No. L4391), blank control (control), 5%CO2,37 DEG C are cultivated 3 days.
2.CpG ODN stimulates spleen t-cell proliferative capacity to analyze
(1) 1 treated cell, 2000rpm/min are collected, room temperature is centrifuged 5 minutes.Collect cells and supernatant, measurement The cell factor of secretion, -80 DEG C of preservation are spare.Cell precipitation is washed 1 time with DPBS, cell count, every 100 μ L 1 × 10 of pipe6 A cell.Suitable Fc receptor blocking pharmacon (CD16/32) (BD Pharmingen, article No. 553142) is added in every pipe, and room temperature is protected from light It is incubated for 5 minutes.
(2) suitable antibody CD3 (BD Pharmingen, article No. 553063) and corresponding isotype control Ab (BD is added Pharmingen, article No. 553925), setting negative cells compare, and 4 DEG C are protected from light incubation 30min.With DPBS (EDTA 2mmol+ 1% serum) washing 1 time, 400 μ L DPBS are added.
(3) flow cytometer (BD Accrri C6Plus) is analyzed
It is charted divided by the multiple of the average value of blank control group (fold differences=reality using the average value of experimental group data Test cell mean/blank control cell mean), all tests at least repetitive operation 3 times.
As a result as shown in Figure 1, compared with other groups including positive control LPS, CpG-45-mod has stronger stimulation T The ability of proliferation, CpG-45 that as a result illustrate synthesis, thio-modification have the potentiality as immunopotentiator or immunologic adjuvant.
3.CpG ODN stimulates splenic B cells proliferative capacity to analyze
(1) 1 treated cell, 2000rpm/min are collected, room temperature is centrifuged 5 minutes.Collect cells and supernatant, measurement The cell factor of secretion, -80 DEG C of preservation are spare.Cell precipitation is washed 1 time with DPBS, cell count, every 100 μ L 1 × 10 of pipe6 A cell.Suitable Fc receptor blocking pharmacon (CD16/32) (BD Pharmingen, article No. 553142) is added in every pipe, and room temperature is protected from light It is incubated for 5 minutes.
(2) suitable antibody CD19 (BD Pharmingen, article No. 550992) and corresponding isotype control Ab is added (BD Pharmingen, article No. 553932), setting negative cells compare, and 4 DEG C are protected from light incubation 30 minutes.With DPBS (EDTA It 2mmol+1%FBS) washs 1 time, 400 μ L DPBS is added.
(3) flow cytometer (BD Accrri C6Plus) is analyzed
It is charted divided by the multiple of the average value of blank control group (fold differences=reality using the average value of experimental group data Test cell mean/blank control cell mean), all tests at least repetitive operation 3 times.
As a result as shown in Fig. 2, compared with other groups including positive control LPS, CpG-45-mod has very strong stimulation B The ability of proliferation, CpG-45 that as a result illustrate synthesis, thio-modification have the potentiality as immunopotentiator or immunologic adjuvant.
4.CpG ODN stimulates spleen cell secrete cytokines capability analysis
Cytokines measurement uses the kit LEGENDplex of Biolegend companyTM Mouse Th Cytokine Panel (13-plex) (Biolegend, article No. 740005)
(1) preparation of samples: the cells and supernatant that (1) obtains in above-mentioned 2 or 3 is diluted as sample assay buffer One times (+50 μ l assay buffer of 50 μ l sample).
(2) system is prepared:
1) add 25ul Assay Buffer to various kinds quality control.
2) add 25ul each sample to respective sample pipe.
3) add each standard items of 25ul to respective standard quality control.
4) add 25ul mixing beads to each pipe (sufficient vortex is just vortexed 1 time after 2-3 minutes every).
5) when adding Beads, Beads is avoided to settle, the final volume of every pipe is 75ul.
(3) it is protected from light, 310rpm/min shakes EP pipe, is incubated at room temperature 2 hours.
(4) directly add 25ul SA-PE to each pipe.
(5) it is protected from light, 310rpm/min shakes EP pipe, is incubated at room temperature 30 minutes.
(6) 1000g is centrifuged 5 minutes.
(7) it is carefully inhaled with pipette tips and abandons 125 μ l of supernatant.Pay attention to not absorbing Beads, is eliminated as much as liquid.
(8) every pipe adds 200ul 1*WB.Vortex Beads, 1000g centrifugation 5 minutes, abandon supernatant.
(9) every pipe adds 200-300ul 1*WB.Vortex Beads.
(10) machine testing is analyzed on flow cytometer (BD Accrri C6Plus)
It is examined using Student ' t, there were significant differences when P < 0.05.
As a result fig. 3, it is shown that the stimulation of CpG-45-mod can dramatically increase IL-6, IL-10, IFN-γ and The expression of TNF-α；Do not express IL-2, IL-4, IL-5, IL-9, IL-13, IL-17A, IL-17F, IL-21 and IL-22.Show to close At, the CpG-45 of sulphur band modification can dramatically increase the expression of B cell proliferation relevant cell factor (IL-6 and IL-10).In addition, CpG-45-mod can dramatically increase the expression of IFN-γ, show that it has the immune response of induction Th1 type and resists microorganism intracellular The ability of infection.
The result is consistent with aforementioned stimulation B cell proliferation experimental result, illustrates again, and CpG-45-mod has relatively strong Induction B cell differentiation and proliferation ability.
The application in mouse is being immunized as adjuvant joint aftosa inactivation antigen in embodiment 2, CpG-45-mod
1, animal
SPF grades of BALB/c (zoopery factory, Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), every group 10, grouping is such as Under:
Table 2
Group Adjuvant, antigen component Size of animal (only)
1(CpG-45-mod) CpG-45-mod, FMDV Ag, 206 adjuvants 10
2(206) FMDV Ag, 206 adjuvants 10
3(PBS) PBS 10
2, vaccine formulation:
Vaccine needed for group 1: by CpG-45-mod, FMDV Ag (Foot and Mouth Disease Virus, FMDV) (foot and mouth disease virus) (offer of middle peasant Witter Biotechnology Co., Ltd), 206 adjuvants are dissolved in PBS, obtain vaccine, and CpG- 45 concentration is 10 μ g/100 μ l, the concentration of FMDV Ag is 5 μ g/100 μ l, the concentration of 206 adjuvants is that 50% (volume basis contains Amount).
Vaccine needed for group 2: FMDV Ag, 206 adjuvants are dissolved in PBS, obtain vaccine, and the concentration of FMDV Ag is 5 μ g/100 μ l, 206 adjuvants concentration be 50% (volume ratio).
Vaccine needed for group 3: the PBS of 0.01M, pH7.2.
3, vaccine emulsify: prepared vaccine, pushed back with two 50mL syringes beat, mix 15 minutes, be placed in 4 DEG C to With.
4, animal immune: the vaccine that will have been emulsified, two back leg leg muscle of intramuscular injection, every 100 μ l of leg, every mouse 200 μ l vaccines are immunized altogether.
5, in immune different time points, the 0th, 7,14,28,60,90 day, 200 serum collection: are acquired from tail vein respectively μ l blood.4 DEG C overnight, and 4 DEG C of 4000rpm/min are centrifuged 15 minutes, collects supernatant, and extremely -80 DEG C of preservation spare.
6, antibody determination: Liquid-phase blocking ELISA (Liquid-phase blocking sandwich ELISA, LB- ELISA) (middle peasant Witter Biotechnology Co., Ltd, article No. A10171), specific as follows:
1) preparation of reagent: with cleaning solution, the coating buffer, substrate solution of the material preparation experiment that kit provides.
2) it is coated with elisa plate: with coating buffer with 1:1000 dilution rabbit anti-serum IgG to working concentration, to elisa plate In every hole add 50 μ l solution, sealing plate film sealing plate ambient temperature overnight after concussion 2-3 minutes.
3) 50 μ l/ hole dosages antigen-antibody reaction (control serum and serum to be checked): are pressed on U-shaped blood-coagulation-board by blood to be checked A clear times serial dilution of opposing, and by the dilution of yin and yang attribute control serum progress proper proportion, it is diluted to PBST using concentration Viral antigen (1:8), then 50 μ l are added in every hole, and viral antigen control wells add 100 μ l.Oscillation mixes, sealing plate, and 4 DEG C overnight.
4) it is washed elisa plate 5 times with washing buffer PBST, is dried on blotting paper, antigen-antibody reaction plate takes out from 4 DEG C After be placed at room temperature for 5 minutes, each hole virus and serum mixture are successively transferred on elisa plate, every hole is added 50 μ l, sealing plate, 37 DEG C are incubated for 1 hour.
5) it is equally washed elisa plate 5 times with washing buffer PBST, cavy is diluted with 1:1000 with guinea pig antiserum dilution Antiserum is to working concentration, and every hole is added 50 μ l, sealing plate, 37 DEG C incubation l hours.
6) after being same as above board-washing, rabbit-anti cavy enzyme conjugates is diluted to working concentration with 1:500 with PBST, 50 μ are added in every hole L, 37 DEG C are incubated for 1 hour.
7) after board-washing, 50 μ l substrate solutions are added in every hole, and 37 DEG C are incubated for 15 minutes.
8) every hole adds 50 μ l terminate liquids to terminate reaction, reads absorbance value under 492nm wavelength immediately.
9) result judgement: 50% with viral antigen control mean OD value is critical value, is detected serum OD value greater than critical The hole of value is negative hole, is positive hole less than or equal to critical value, and the OD value in positive hole is equal to corresponding dilute when critical value Degree of releasing is the antibody titer of this part of serum.If critical value is between two titres, antibody titer takes median.
It is examined using Student ' t, there were significant differences when P < 0.05.
Antibody titer measurement result is as shown in Figure 4, it can be seen that the antibody titer of CpG-45-mod group is significantly larger than 206 Group, and the duration of CpG-45-mod group antibody remains within higher titre after 90 days, illustrates that CpG-45 can be non-specific The antibody titer of the enhancement antigen of property.
Therefore, the CpG-45 that the present invention synthesizes has strong immune-enhancing activity, can be used as immunopotentiator or immune Adjuvant is added in animal vaccine product, and the immune response for capableing of enhancement antigen is horizontal, is reached prevention and is controlled the mesh of epidemic disease 's.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of CpG oligonucleotide sequences and its application with immune-enhancing activity
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
tccatgacgt tcctgacgtt g 21

Claims (7)

1. a kind of oligodeoxynucleotide, nucleotides sequence is classified as sequence 1, and all nucleotide are equal in the oligodeoxynucleotide For thio-modification.
2. oligodeoxynucleotide described in claim 1 is in the application as immunopotentiator or immunologic adjuvant.
3. application of the oligodeoxynucleotide described in claim 1 in the immunogenicity for improving antigen or vaccine.
4. oligodeoxynucleotide described in claim 1 is preparing the application in vaccine.
5. a kind of vaccine comprising immunogene and oligodeoxynucleotide of any of claims 1 or 2.
6. vaccine described in application according to claim 3 or 4 or claim 5, it is characterised in that:
The vaccine is animal vaccine.
7. vaccine described in application according to claim 3 or 4 or claim 5, it is characterised in that: the vaccine is Object vaccine, the animal vaccine are aftosa vaccine.
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