CN102212524A - Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant - Google Patents
Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant Download PDFInfo
- Publication number
- CN102212524A CN102212524A CN2010101381623A CN201010138162A CN102212524A CN 102212524 A CN102212524 A CN 102212524A CN 2010101381623 A CN2010101381623 A CN 2010101381623A CN 201010138162 A CN201010138162 A CN 201010138162A CN 102212524 A CN102212524 A CN 102212524A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- single chain
- adjuvant
- described single
- chain deoxynucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to oligodeoxyribonucleotide used as the immunomodulator of cattle and pig and the vaccine adjuvant. CpG ODN has very high potential application value in various aspects such as the aspect of being used as the vaccine adjuvant, the aspect of resisting microbial infection and the aspect of treating tumor, which can be well verified in human and mouse. The immunostimulation effect of CpG ODN has species specificity. The oligodeoxyribonucleotide can effectively help the foot-and-mouth disease recombinant protein vaccine generate a neutralizing antibody and be used to prepare cattle and pig immunomodulators and vaccine adjuvants.
Description
Technical field:
The present invention relates to a kind of deoxy-oligonucleotide and as the purposes of ox, pig immunomodulator and vaccine adjuvant
Background technology:
Oligodeoxynucleotide (the CpG oligodeoxynucleotide that contains cytosine(Cyt)-guanine dinucleotides motif of synthetic, CpGODN) can stimulate the natural immune system of higher animal, immunocytes such as activating B cell, scavenger cell (M φ), dendritic cell (DC) and NK cell, and induce the secretion of cytokines such as IL-2, IL-6, IL-12, IFN-γ.CpG ODN is as vaccine adjuvant, and all many-sides such as opposing infected by microbes and treatment tumour have very high potential using value, are well verified on people and mouse.
Studies show that the immunostimulation of CpG ODN has species specificity, promptly, then may not show biological activity same or same intensity another kind of animal or human to the bioactive CpGODN of a kind of animal performance performance.Therefore, the specific CpG ODN at every kind of animal still needs purposive research.That the present invention relates to is the CpG ODN that activation all can take place the immunocyte of ox, pig, with as pig, ox immunomodulator and vaccine adjuvant.
Summary of the invention
In order to seek all effective CpG ODN of ox, pig, with its immunomodulator and vaccine adjuvant as pig, ox, designed multiple CpGODN, having detected it stimulates the activity of ox, pig, mouse immune cell proliferation, and has detected its effect as foot and mouth disease recombinant protein vaccine adjuvant in mouse and ox.The result shows, RW03 (the CpG ODN that the present invention is disclosed, its sequence is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ') have a following effect:
1, ox, pig, mouse immune cell had the effect of stimulating proliferation significantly;
2, in the mouse body in the experiment, RW03 can effectively assist foot and mouth disease recombinant protein vaccine generation neutrality antibody;
3, in the ox body in the experiment, RW03 can effectively assist the foot and mouth disease recombinant protein vaccine to produce neutrality antibody, and protects ox to make it to resist the attack of foot and mouth disease virus.
These effects make RW03 can be used to prepare ox and pig immunomodulator and vaccine adjuvant.
Research involved in the present invention mainly is:
1, RW03 is to the research of ox immunocyte immunostimulation
The splenocyte that we stimulate ox with CpG ODN, by
3The H-TdR method detects the propagation situation of cell, at the stimulating activity of vitro detection CpG ODN to the ox immunocyte.The result shows: RW03 can stimulate the propagation of ox splenocyte effectively, and its effect is dose-dependently, and stimulus intensity is better than CpG ODN 2006.Illustrate that RW03 is a kind of CpG ODN of ox sensitivity, can be used to prepare immunomodulator and the vaccine adjuvant of ox, can use its immune effect of enhancing together with the vaccine of ox.
2, RW03 is to the research of pig immunocyte immunostimulation
Stimulate the pig peripheral blood mononuclear cell with CpG ODN, by
3The H-TdR method detects the propagation situation of cell, at the stimulating activity of vitro detection CpG ODN to the pig immunocyte.The result shows: RW03 can stimulate the propagation of pig spleen cell effectively, and its stimulus intensity is compared indifference with CpG ODN2006.Illustrate that RW03 is the CpG ODN of a boar sensitivity, can be used to prepare immunomodulator and the vaccine adjuvant of pig, can use its immune effect of enhancing together with the vaccine of pig.
3, RW03 is to the research of mouse immune cellular immunization hormesis
We stimulate the mouse boosting cell proliferation experiment with CpG ODN, by
3The H-TdR method detects the propagation situation of cell, at the stimulating activity of vitro detection CpGODN to the mouse immune cell.Bibliographical information, CpG ODN 1826 can stimulate the propagation of mouse immune cell significantly, with its positive control as experiment.The result shows: RW03 can stimulate mouse boosting cell propagation significantly, compares indifference with CpG ODN 1826.Illustrate that RW03 also is a kind of CpG ODN of mouse sensitivity, can adopt mouse to carry out its immune effect of experimental verification in the body as pig, ox immunomodulator and vaccine adjuvant.
3, RW03 is as the adjuvant of foot and mouth disease recombinant protein vaccine
Respectively with RW03 and aluminium or 206 oily combined utilization, as the adjuvant immunity mouse of foot and mouth disease recombinant protein vaccine.Utilize indirect ELISA method to detect neutralizing antibody level in the serum.The result shows: RW03 and aluminium or 206 oil are united the adjuvant as the foot and mouth disease recombinant protein vaccine, can produce high-caliber foot and mouth disease virus specific antibody in the mouse body.
Respectively with RW03 and aluminium or 206 oily combined utilization, as the adjuvant immunity ox of foot and mouth disease recombinant protein vaccine.The immunostimulant that utilizes ELISA method, cell neutralization test, suckling mouse neutralization test and foot and mouth disease virus challenge test to detect RW03 is renderd a service.The result shows: RW03 can assist the foot and mouth disease recombinant protein vaccine to produce high-caliber foot and mouth disease virus neutralizing antibody at ox body internal stimulus, and the protection ox makes it to resist the attack of foot and mouth disease virus.
Term in the invention:
" oligonucleotide ": oligonucleotide is by sugar (as ribodesose or ribose), the mononucleotide of phosphate group and based composition is connected and the molecule that forms, wherein glycan molecule and base connect into nucleosides (nucleoside), nucleosides is connected to form Nucleotide (nucleotide) by phosphate group, the base that forms nucleosides has pyrimidine and purine, pyrimidine has thymus pyrimidine (thymine, be abbreviated as T or t) and cytosine(Cyt) (cytosine, be abbreviated as C or c), purine has VITAMIN B4 (adenine, be abbreviated as A or a) and guanine (guanine is abbreviated as G or g).Oligonucleotide can be strand also can be double-stranded.In the present invention, " oligonucleotide " (Oligodeoxynucleotide ODN) can replace with its english abbreviation ODN.
" chemically modified ": compare with the DNA of nature, the oligonucleotide among the present invention can be through various chemically modifieds, and the position of modification can occur in the phosphodiester bond between the nucleosides, ribose units is or/and organic base (A, T, C, G, uridylic, uridine is abbreviated as U or u).Can modify between the synthesis phase of oligonucleotide or after synthetic.Chemically modified between synthesis phase can be modified at the inside or the 5` end of oligonucleotide.Oligonucleotide after synthetic can but be not limited to carry out chemically modified at active group (as the phosphoric acid or the hydroxyl of 5` or 3` end).The professional can understand the concrete mode of these chemically modifieds.Chemically modified among the present invention comprises the backbone modification of oligonucleotide.Wherein, the non-bridge phosphoric acid Sauerstoffatom at least one internucleotide linkage is replaced by sulphur atom.Nonionic DNA analogue also can take place in the skeleton of oligonucleotide, modify as alkyl, fragrance-phosphonate (charged phosphonate Sauerstoffatom is replaced by alkyl, aromatic base), the part of the Sauerstoffatom in phosphodiester and the alkyl phosphotriester is modified by alkylation for another example.Oligonucleotide can also be the mosaic of phosphorothioate and phosphodiester.Chemically modified comprises that also base substitutes, and substitutes as C-5 propine pyrimidine and the alternative purine of 7-deaza-7.Chemically modified also comprises base modification.The base of base in chemically being different from typical nature of modifying, but have their basic chemical structures.Oligonucleotide among the present invention can also be modified with cytosine derivative or thymidine derivative.Here cytosine derivative is meant cytosine(Cyt) sample nucleosides (except cytosine(Cyt)).The thymidine derivative is meant the phonetic sample nucleosides of thymus gland (not comprising thymus pyrimidine).In addition, the modification of the oligonucleotide among the present invention can be two or dibasic alcohol of a terminal connection at oligonucleotide, as TEG or six ethylene glycol.
CpG:C represents cytosine(Cyt), and G represents guanine, and p represents phosphodiester bond.CpG is the non-methylated dinucleotides that is connected into by phosphodiester bond by cytosine(Cyt) and guanine.
" adjuvant ": be some prior to antigen or simultaneously with the material of antigen hybrid injection animal, can play the immunogenic effect of enhancement antigen.In animal vaccine, adjuvant can strengthen its immune efficacy.
Description of drawings:
Fig. 1: oligonucleotide (ODN) is to the hormesis of bull ox splenocyte propagation
Shown in the X-coordinate is title and the ODN blank of ODN, and the ODN final concentration is 5 μ g/ml; Ordinate zou is a stimulation index, and promptly CpG ODN stimulates ox splenocyte ratio of the radioactivity reading (cpm) of per minute and the radioactivity reading of ODN blank in every hole after 72 hours.8 oxen are represented as the shape that is indicated among the figure with 8 kinds respectively.The result shows, compares with ODN blank group and 2006 control groups, and RW03 can stimulate ox splenocyte propagation (P＜0.01) significantly.
Fig. 2: oligonucleotide (ODN) is to the amount effect curve of the ox splenocyte effect of stimulating proliferation
X-coordinate is the dosage of the ODN that adds in the experiment, and ordinate zou stimulates ox splenocyte radioactivity reading (cpm) of per minute in every hole after 72 hours for CpG ODN, and two kinds of oligonucleotide and ODN blank are respectively with representing as the shape that is indicated among the figure.The result shows, compares with blank group and 2006 control groups, and RW03 can stimulate ox splenocyte propagation significantly, and its hormesis strengthens (P＜0.01) along with the increase of dosage.
Fig. 3: oligonucleotide (ODN) is to the hormesis of pig peripheral blood mononuclear cell propagation
Shown in the X-coordinate is title and the ODN blank of ODN, and ordinate zou stimulates pig PBMC radioactivity reading (cpm) of per minute in every hole after 72 hours for CpG ODN.3 pigs are represented as the shape that is indicated among the figure with 3 kinds respectively.The result shows, compares with ODN blank group, and RW03 can stimulate pig peripheral blood mononuclear cell propagation (P＜0.01) significantly.
Fig. 4: oligonucleotide (ODN) is to the hormesis of mouse boosting cell propagation
Shown in the X-coordinate is title and the ODN blank of ODN, and ordinate zou stimulates mouse boosting cell radioactivity reading (cpm) of per minute in every hole after 72 hours for CpG ODN.3 mouse are represented as the shape that is indicated among the figure with 3 kinds respectively.The result shows, compares with ODN blank group, and RW03 can stimulate mouse boosting cell propagation (P＜0.01) significantly.
Fig. 5: oligonucleotide (ODN) and the adjuvant of aluminium adjuvant coupling as the foot and mouth disease recombinant protein vaccine
X-coordinate is a fate, and ordinate zou is an antibody titer.Three kinds of immunization wayses are respectively with representing as the shape that is indicated among the figure.The result shows: RW03 and aluminium are united as behind the foot and mouth disease recombinant protein vaccine adjuvant, compare with group with the vaccine list and produce the antibody time more early, produce antibody horizontal higher (P＜0.01).
Fig. 6: oligonucleotide (ODN) and the adjuvant of 206 oily adjuvant couplings as the foot and mouth disease recombinant protein vaccine
X-coordinate is a fate, and ordinate zou is that microplate reader detects A
492Numerical value is represented antibody horizontal.Three kinds of immunization wayses are respectively with representing as the shape that is indicated among the figure.The result shows: RW03 and 206 oil are united as behind the foot and mouth disease recombinant protein vaccine adjuvant, compare with group with the vaccine list and produce antibody horizontal higher (P＜0.01).
The detection of FMDV neutralizing antibody in the bovine serum of Fig. 7 immunity (in the BHK-21 cell and experiment)
Use A7+206 oil, the foot and mouth disease virus vaccine immune cattle of A7+206 oil+RW03 or deactivation, by way of being intramuscular injection (i.m), the time of immunity is the 0th day, the blood samplings in the 14th, 28,56 and 84 days after the 0th day and immunity.Detect foot-mouth disease poison specific antibody with the foot and mouth disease virus specific ELISA.The mean value of each symbology antibody horizontal is with geometric mean titer ± standard deviation (GMT ± S.D.) expression.Every on behalf of dynamic antibody, curve produce.The dosage of RW03 is 500 μ g/ oxen.
*P＜0.05。
The sequence of RW03 is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1).
After attacking, Fig. 8 foot and mouth disease virus respectively organizes the period of disease of ox
Use A7+206 oil, the foot and mouth disease virus vaccine immune cattle of A7+206 oil+RW03 or deactivation is by way of being intramuscular injection (i.m).Niu Jing to immunity penetrates tongue injection lethal quantity foot and mouth disease virus FMDV), attacking ten days records of poison back observation incidence.
The sequence of RW03 is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1).
Embodiment
Embodiment 1RW03 is to the hormesis of bull ox splenocyte propagation
1, instrument, equipment, equipment, reagent and material:
Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, 200 mesh filter screens, the dropper of all size etc., β calculating instrument, glass fiber filter paper etc.
ODN: synthetic by Takara company, the OPC rank, its phosphoric acid skeleton is full thio-modification except that special indicating.With PBS (prescription is with reference to " molecular cloning experiment guide (third edition) ") dissolving, ultraviolet spectrophotometer is quantitative.
Erythrocyte cracked liquid: take by weighing KHCO respectively
31g, EDTA.Na
20.37g, NH
4Cl 8.29g adds 800ml water, transfers pH value to 7.2～7.4, adds water to 1L again, filtration sterilization.
IMDM nutrient solution: IMDM 17.7g (GIBCO), sodium bicarbonate 2.0 gram, gentamicin 100,000 units add ultrapure water to 1000 milliliter, 0.22 micron filter membrane suction filtration degerming, packing.
The IMDM substratum that contains 10% foetal calf serum: 10 milliliters of foetal calf serums (Invitrogen), 90 milliliters of IMDM substratum.
3H-thymidine: Shanghai Atomic Nucleus Inst., Chinese Academy of Sciences.
Scintillation solution: take by weighing 2 (PPO) 4 gram, 100 milligrams of 1,4 pair of [the 5-Ben Ji oxazolyl-2] benzene are dissolved in 1000 milliliters of dimethylbenzene.
Fresh bovine spleen: take from the healthy calf of 8 1-1.5 (two positive slaughterhouses, Changchun provide).
The ODN that adopts is RW03 and 2006.The 2006th, the CpG ODN to ox immunocyte sensitivity of bibliographical information is as positive control.(R.A.Pontarollo，et?al. Monocytes?are?required?for?optimum?in?vitro?stimulation?of?bovine?peripheral?bloodmononuclear?cells
2006：5’-tcgtcgttttgtcgttttgtcgtt-3’。
RW03：5′-tcgcgaacgttcgcccgatcgtcggta-3’。
2, method:
From the aseptic ox spleen that obtains, take out a little spleen tissue and place on the obscure glass, grind the pressure spleen, filter through 200 mesh filter screens and obtain cell suspension.Level centrifugal (1,000g, 5 minutes) is abandoned supernatant, behind the re-suspended cell, add the 1ml erythrocyte cracked liquid, room temperature is placed 1min, adds the IMDM to 5ml of 10% foetal calf serum, again sub-level centrifugal (1,000g, 5 minutes), resuspended after, 200 mesh filter screens filter splenocyte suspension, cell counting.
Adopt culture plate at the bottom of 96 hole circles, cultivate ox splenocyte, 2 * 10 with the IMDM that contains 10% foetal calf serum
5Individual cells/well.The splenocyte of every ox is all set up experimental group, control group and ODN blank group.Experimental group adds RW03, and control group adds 2006, and ODN blank group adds the IMDM nutrient solution that contains 10% foetal calf serum.Each final concentration of organizing ODN in the culture hole is 5 μ g/ml respectively, establishes 3 multiple holes.
Each is organized splenocyte and, adds after 54 hours in the ODN effect
3H-Tdr, 0.5 μ Ci/ hole, after 18 hours, collecting cell 50 ℃, was dried on glass fiber filter paper in 3 hours.The corresponding glass fiber filter paper is joined in the scintillation solution, after 12 hours, detect the radioactivity reading (cpm) of per minute with the β calculating instrument.The gained data are learned processing by statistics, and wherein stimulation index SI is the ratio of experimental group or control group and ODN blank group cpm.
3, experimental result:
Compare with ODN blank group, RW03 can stimulate ox splenocyte propagation (P＜0.01) significantly; Compare with control group 2006, RW03 also shows stronger stimulation ox splenocyte proliferation function (P＜0.01), the results are shown in Figure 1.
Conclusion: RW03 can stimulate the ox immune cell propagation significantly, has stronger immunostimulation than the Newt opposite sex oligonucleotide of having found 2006.
Embodiment 2RW03 stimulates the dose curve of the hormesis of ox splenocyte propagation
1, instrument, equipment, equipment, reagent and material:
With embodiment 1.
2, method:
From the aseptic ox spleen that obtains, take out a little spleen tissue and place on the obscure glass, grind the pressure spleen, filter through 200 mesh filter screens and obtain cell suspension.Level centrifugal (1,000g, 5 minutes) is abandoned supernatant, behind the re-suspended cell, add the 1ml erythrocyte cracked liquid, room temperature is placed 1min, adds the IMDM to 5ml of 10% foetal calf serum, again sub-level centrifugal (1,000g, 5 minutes), resuspended after, 200 mesh filter screens filter splenocyte suspension, cell counting.
Adopt culture plate at the bottom of 96 hole circles, cultivate ox splenocyte, 2 * 10 with the IMDM that contains 10% foetal calf serum
5Individual cells/well.Set up experimental group, control group and ODN blank group, experimental group adds ODN RW03, and control group adds ODN 2006, and ODN blank group adds the IMDM nutrient solution that contains 10% foetal calf serum.The final concentration of ODN is 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml in every group of culture hole, and each concentration is all established 3 multiple holes in each group.
After the ODN effect 54 hours, add
3H-Tdr, 0.5 μ Ci/ hole, after 18 hours, collecting cell 50 ℃, was dried on glass fiber filter paper in 3 hours.The corresponding glass fiber filter paper is joined in the scintillation solution, after 12 hours, detect the radioactivity reading (cpm) of per minute with the β calculating instrument.The gained data are learned processing by statistics.
3, result:
Compare with ODN blank group, RW03 can stimulate ox splenocyte propagation (P＜0.01) significantly; Compare with control group 2006, RW03 also shows stronger stimulation ox splenocyte proliferation function, and its effect of stimulating proliferation strengthens (P＜0.01) with dosage, the results are shown in Figure 2.
Conclusion: RW03 can stimulate the ox immune cell propagation significantly, and has tangible dose-effect relationship.Has stronger immunostimulation than the Newt opposite sex oligonucleotide of having found 2006.
Embodiment 3RW03 is to the hormesis of pig PBMC propagation
1, instrument, equipment, equipment, reagent and material
Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, 200 mesh filter screens, the dropper of all size etc., β calculating instrument, glass fiber filter paper etc.
ODN: synthetic by Takara company, the OPC rank, its phosphoric acid skeleton is full thio-modification except that special indicating.With PBS (prescription is with reference to " molecular cloning experiment guide (third edition) ") dissolving, ultraviolet spectrophotometer is quantitative.
3H-thymidine: Shanghai Atomic Nucleus Inst., Chinese Academy of Sciences.
Scintillation solution: take by weighing 2 (PPO) 4 gram, 100 milligrams of 1,4 pair of [the 5-Ben Ji oxazolyl-2] benzene are dissolved in 1000 milliliters of dimethylbenzene.
The pig peripheral blood of anticoagulant heparin: 5 monthly ages, healthy landrace.
Poly-glucose-urografic acid methylglucamine salt: proportion 1.077 ± 0.001, Beijing ancient cooking vessel state biotech company.
IMDM nutrient solution: IMDM 17.7g (GIBCO), sodium bicarbonate 2.0 gram, gentamicin 100,000 units add ultrapure water to 1000 milliliter, 0.22 micron filter membrane suction filtration degerming, packing.
The IMDM substratum that contains 10% foetal calf serum: 10 milliliters of foetal calf serums (Invitrogen), 90 milliliters of IMDM substratum.
The ODN that adopts is RW03 and 2006.2006 are meant CpG2006 (5 '-tcgtcgttttgtcgttttgtcgtt-3 ') (Robert Rankin, et al.CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highlyconserved.Antisense nucleic A, 2001, Vol 11:333-340).
Used oligonucleotide sequence is as follows in the present embodiment:
2006：5’-tcgtcgttttgtcgttttgtcgtt-3’。
RW03：5′-tcgcgaacgttcgcccgatcgtcggta-3’。
2, method:
Separate the mononuclearcell of pig peripheral blood with poly-glucose-urografic acid methylglucamine salt lymphocyte separation medium.Parting liquid and heparin anti-coagulating volume ratio are 2: 1, and level centrifugal (1,000 * g, 30 minutes) with the liquid band of dropper absorption mononuclearcell, is inserted in another centrifuge tube, adds 5 milliliters of serum free mediums, 1, and centrifugal 10 minutes of 000g abandons supernatant.Repeated washing twice is with 5 milliliters of IMDM substratum re-suspended cells that contain 10% foetal calf serum, cell counting.
Adopt culture plate at the bottom of 96 hole circles, cultivate pig peripheral blood mononuclear cell, 5 * 10 with the IMDM nutrient solution that contains 10% foetal calf serum
5Individual cells/well.Set up experimental group, control group and ODN blank group, experimental group adds ODN RW03, and control group adds ODN 2006, and ODN blank group adds the IMDM nutrient solution that contains 10% foetal calf serum.The final concentration of ODN is 3 μ g/ml in every group of culture hole, establishes 3 multiple holes.
After the ODN effect 54 hours, add
3H-Tdr, 0.5 μ Ci/ hole remakes with after 18 hours, collecting cell on glass fiber filter paper, 50 ℃ of oven dry in 3 hours.Corresponding glass paper is joined in the scintillation solution, act on after 12 hours, detect 1 minute radioactivity reading (cpm) with the β calculating instrument.
3, experimental result:
Compare with ODN blank group, RW03 can stimulate pig PBMC propagation (P＜0.01) significantly; Compare indifference (P＞0.05) with control group 2006, the results are shown in Figure 3.
Conclusion: RW03 can effectively stimulate the activation and proliferation of pig immunocyte.
Embodiment 4RW03 is to the outgrowth hormesis of mouse boosting cell
1, instrument, equipment, equipment, reagent and material:
With embodiment 1.
BALB/c mouse: Bethune medical college of Jilin University experimentation on animals center.
The ODN that adopts is RW03 and 1826.CpG ODN 1826 is meant (Heidi H.van Ojik, et al.CpG-A and BOligodeoxynucleotides Enhance the Efficacy of Antibody Therapy by Activating Different Effector CellPopulations.Cancer Research, 1 Sep, 2003, Vol 63,5595-5600).
Used oligonucleotide sequence is as follows in the present embodiment:
The sequence of RW03 is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 '.
1826 sequence is: 5 '-tccatgacgttcctgacgtt-3 ';
2, method:
After mouse was put to death in dislocation, aseptic separating spleen was placed spleen on the obscure glass, grinds the pressure spleen, filtered through 200 mesh filter screens and obtained cell suspension.Level centrifugal (1,000g, 5min), abandon supernatant, behind the re-suspended cell, add the 1ml erythrocyte cracked liquid, room temperature is placed 1min, add the RPMI1640 to 5ml of 10% foetal calf serum, again sub-level centrifugal (1,000g, 5 minutes), after resuspended, 200 mesh filter screens filter the mouse boosting cell suspension, cell counting.
Adopt culture plate at the bottom of 96 hole circles, to contain the IMDM culture medium culturing mouse boosting cell of 10% foetal calf serum, 6 * 10
5Individual cells/well.Set up experimental group, control group and ODN blank group, experimental group adds ODN RW03, and control group adds ODN 1826, and ODN blank group adds the IMDM nutrient solution that contains 10% foetal calf serum.The final concentration of ODN is 3 μ g/ml in every group of culture hole, establishes 3 multiple holes.
After the ODN effect 54 hours, add
3H-Tdr, 0.5 μ Ci/ hole, after 18 hours, collecting cell 50 ℃, was dried on glass fiber filter paper in 3 hours.The corresponding glass fiber filter paper is joined in the scintillation solution, after 12 hours, with 1 minute radioactivity reading (cpm) of β calculating instrument detection.
3, experimental result:
Compare with ODN blank group, RW03 can stimulate mouse boosting cell propagation significantly, compares indifference (P＞0.05) with control group 1826, the results are shown in Figure 4.
Conclusion: RW03 can effectively stimulate the activation and proliferation of mouse boosting cell.
Embodiment 5RW03 and aluminium adjuvant are united the experiment as foot and mouth disease recombinant protein vaccine adjuvant
1, instrument, equipment, equipment, reagent and material:
Syringe, blade, hemospast, incubator, cryogenic refrigerator, low-temperature and high-speed whizzer, sample injector, microplate reader, enzyme mark bar, the Ep pipe of all size etc.
Foot and mouth disease virus recombinant protein vaccine A7: molecular biology teaching and research room of Bethune medical college of Jilin University makes up and expression and purification, and the hostis pecoris recombinant protein vaccine (A7) that studies have shown that Inner Mongol Jinyu Group is protected clever biologics company limited can induce protective immunological reaction at foot and mouth disease virus at the ox body.
Aluminium hydroxide gel physiological saline diluent: Heilongjiang Province's biological products one factory.
PBS: prescription is with reference to " molecular cloning experiment guide (third edition) ".
Coating buffer: Na
2CO
31.95g, NaHCO
32.93g pH value to 9.6 is transferred in ultrapure water 800ml dissolving, is settled to 1000ml.
Confining liquid: 5ml FBS+95ml PBS
Washings: 0.5ml polysorbas20+999.5ml PBS.
Substrate solution: 10 * the first and second liquid 1ml (0.1M citric acid, 0.2M Na
2HPO
4), ultrapure water 9ml, 30%H
2O
215 μ l, OPD 4mg.
The ox Asia I type foot and mouth disease virus and the positive serum of deactivation: Inner Mongol Jinyu Group is protected clever biologics company limited.
Cavy: 250-300g, Bethune medical college of Jilin University experimentation on animals center.
The ODN that adopts is RW03.Used oligonucleotide sequence is as follows in the present embodiment:
RW03：5′-tcgcgaacgttcgcccgatcgtcggta-3’。
2, method:
These section office are made up the also foot and mouth disease virus recombinant protein vaccine A7 immune guinea pig of expression and purification voluntarily, be divided into 4 groups, 4 every group, the hindlimb muscle injection.Experiment is grouped as follows: the A7 protein groups, and A7200ug/ only adds isopyknic PBS; A7 albumen+aluminium group, A7200ug/ only adds isopyknic aluminium adjuvant; A7 albumen+RW03+ aluminium group, A7200ug/ only adds isopyknic aluminium adjuvant, adds 20ugRW03.The immunity time: carried out immunity first time on the 1st day, it is immune to carry out the second time on the 36th day.
Immunity a few days ago tail vein is got blood as negative serum, gets blood after the first immunisation weekly, is positioned in the test tube.37 ℃ 15 minutes.Tilt to place for 4 ℃ and spend the night, treat that serum is fully separated out after, collect serum, aseptic subpackaged ,-20 ℃ of preservations.Be used to be ELISA.
Coating buffer mixes with the ox Asia I type hoof-and-mouth disease venom equal-volume of deactivation, every hole 100 μ l, and 4 ℃ of bags are spent the night.After washings is washed plate 3 times, add confining liquid 200 μ l, placed 2 hours in 37 ℃ of incubators.Washings is washed plate 3 times, and 1: 100 dilution back of serum of collecting after the immunity is added enzyme plate, adds serial dilution simultaneously and divides positive serum, and two multiple holes are established in 100 μ l/ holes, place 1 hour in 37 ℃ of incubators.Washings is washed plate 3 times, adds the anti-cavy IgG of the rabbit antibody of the horseradish peroxidase-labeled of dilution in 1: 5000,100 μ l/ holes, hatched 1 hour for 37 ℃, washings is washed plate 3 times, adds substrate solution, lucifuge was placed 15 minutes, with the reaction of 50ul 2M sulfuric acid color development stopping, surveyed OD value (A with microplate reader
492).With positive control serial dilution degree is ordinate zou, and corresponding OD value is obtained its corresponding extent of dilution by the OD value of each test serum, and conversed tiring of test serum according to tiring of fixed positive serum antibody for the X-coordinate mapping on curve.The results are shown in Figure 5.
3, experimental result:
The result shows, compares with other two groups, and RW03 and aluminium combined utilization improve (p＜0.05) significantly as the adjuvant group antibody horizontal of foot and mouth disease virus recombinant protein vaccine A7.
Conclusion: CpG ODN RW03 and aluminium adjuvant coupling can be effectively as the adjuvant of foot and mouth disease recombinant protein vaccine.
1, instrument, equipment, equipment, reagent and material:
Syringe, blade, hemospast, incubator, cryogenic refrigerator, low-temperature and high-speed whizzer, sample injector, microplate reader, enzyme mark bar, the Ep pipe of all size etc.
Hostis pecoris recombinant protein vaccine (A7): molecular biology teaching and research room of Bethune medical college of Jilin University makes up and expression and purification, and the hostis pecoris recombinant protein vaccine (A7) that studies have shown that Inner Mongol Jinyu Group is protected clever biologics company limited can induce protective immunological reaction at foot and mouth disease virus at the ox body.
PBS: prescription is with reference to " molecular cloning experiment guide (third edition) ".
Coating buffer: Na
2CO
31.95g, NaHCO
32.93g pH value to 9.6 is transferred in ultrapure water 800ml dissolving, is settled to 1000ml.
Confining liquid: 5ml FBS+95ml PBS
Washings: 0.5ml polysorbas20+999.5ml PBS.
Substrate solution: 10 * the first and second liquid 1ml (0.1M citric acid, 0.2M Na
2HPO
4), ultrapure water 9ml, 30%H
2O
215 μ l, OPD 4mg.
The ox Asia I type foot and mouth disease virus of deactivation: Inner Mongol Jinyu Group is protected clever biologics company limited.
BALB/c mouse: 18-22g, female, Bethune medical college of Jilin University experimentation on animals center.
The ODN that adopts is RW03.Used oligonucleotide sequence is as follows in the present embodiment:
RW03：5′-tcgcgaacgttcgcccgatcgtcggta-3’。
2, method:
Foot and mouth disease virus recombinant protein vaccine A7 immune guinea pig is divided into 3 groups, and 4 every group, the hindlimb muscle injection.Experiment is grouped as follows: the A7 protein groups, and A750 μ g/ only adds isopyknic PBS; A7 albumen+206 oil groups, A750 μ g/ only adds isopyknic 206 oily adjuvants; A7 albumen+206 oil+RW03 group, A750 μ g/ only adds isopyknic 206 oily adjuvants, adds 10 μ g RW03.Carried out immunity on the 0th day.
Immunity a few days ago tail vein is got blood as negative serum, gets blood after the first immunisation weekly, is positioned in the test tube.37 ℃ 15 minutes.Tilt to place for 4 ℃ and spend the night, treat that serum is fully separated out after, collect serum, aseptic subpackaged ,-20 ℃ of preservations.Be used to be ELISA.
Coating buffer mixes with the ox Asia I type hoof-and-mouth disease venom equal-volume of deactivation, every hole 100 μ l, and 4 ℃ of bags are spent the night.After washings is washed plate 3 times, add confining liquid 200 μ l, placed 2 hours in 37 ℃ of incubators.Washings is washed plate 3 times, and 1: 100 dilution back of serum of collecting after the immunity is added enzyme plate, and two multiple holes are established in 100 μ l/ holes, place 1 hour in 37 ℃ of incubators.Washings is washed plate 3 times, adds the anti-cavy IgG of the rabbit antibody of the horseradish peroxidase-labeled of dilution in 1: 5000,100 μ l/ holes, hatched 1 hour for 37 ℃, washings is washed plate 3 times, adds the substrate solution lucifuge and places 15 minutes, with the reaction of 50ul 2M sulfuric acid color development stopping, microplate reader is surveyed OD value (A
492), the results are shown in Figure 6.
3, experimental result
Compare with other two groups, RW03 and 206 oily combined utilization improve (p＜0.05) significantly as the adjuvant group antibody horizontal of foot and mouth disease virus recombinant protein vaccine A7.
Conclusion: CpG ODN RW03 and 206 oily adjuvant couplings can be effectively as the adjuvant of foot and mouth disease recombinant protein vaccine.
In order to verify further whether mixed mode can produce high level and long antibody of extended period in the ox body, we are with its immune cattle.Experiment divides four groups of a PBS control group, A7 albumen+206 oil groups, A7 albumen+206 oil+500 four groups of μ g RW03 and inactivated vaccine groups, every group of 5 oxen, carry out once immunity, in immunity back blood sampling in 14,28,56 and 84 days for the first time, after preparing bovine serum, detect the level of specific antibody in the serum respectively with ELISA, the neutralization of micro-cell, suckling mouse neutral method.
Described material and method are as described in embodiment 5 and 6.A7 albumen is the hostis pecoris recombinant protein vaccine; made up and expression and purification by molecular biology teaching and research room of Bethune medical college of Jilin University, the A7 albumen that studies have shown that Inner Mongol Jinyu Group is protected clever biologics company limited can induce protective immunological reaction at foot and mouth disease virus at the ox body.Inactivated vaccine is the hostis pecoris inactivated vaccine, protects clever biologics company limited by Inner Mongol Jinyu Group and produces.Carried out the ox body in back 84 days in immunity for the first time and attacked the poison experiment, what be used to attack poison is Asia I type foot and mouth disease virus (FMDV).More than experiment is protected clever biologics company limited experimental center at Inner Mongol Jinyu Group and is finished.
(1) specific antibody ELISA detects in the immune cattle serum
In order to detect the neutralizing antibody that whether in the ox body, has induced high titre, exempt to gather in back 28 days and 84 days the bovine serum sample one, detect the level of Asia I type FMDV specific IgG in the serum with the indirect ELISA method of the Asia I C-type virus C bag quilt of deactivation.The indirect ELISA result shows: produced the specific IgG antibody at Asia I type FMDV in the serum of A7 albumen+206 oil+RW03 immune cattle, and be significantly higher than A7+206 oil group and deactivation vaccine group (table 1).
The ELISA of FMDV specific antibody detects in the table 1 immune cattle serum
The RW03 sequence is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1).
(2) provide protection of specificity neutralizing antibody pair cell in the immune cattle serum
For the provide protection of the serum that detects immune cattle pair cell under the condition that homotype Asia I type FMDV attacks, carried out cell virus neutralization test based on the BHK-21 cell.After exempting from the back 14,28,56 and 84 days bovine serums carry out serial dilution and heat inactivation complement one, mix with the Asia I type FMDV that virulence is arranged of an amount of (100TCID50), after carrying out neutralization reaction, add a certain amount of BHK-21 cell, carry out cultivation and the observation of 72h.The result shows: in immunity back 28 days, in the serum of A7 albumen+206 oil+RW03 immune cattle, produced high-caliberly at the specific IgG antibody of Asia I type FMDV, and its antibody horizontal is significantly higher than A7 albumen+206 oil groups (P＜0.01);
In immunity back 84 days, A7 albumen+206 oil+RW03 group antibody horizontal was 2.14 times (figure-7) of inactivated vaccine group.
(3) in the immune cattle serum specificity neutralizing antibody to the provide protection of suckling mouse
For the serum that detects immune cattle under the condition that homotype Asia I type FMDV attacks to the provide protection of suckling mouse, carried out testing based on the virus neutralization of suckling mouse.After back 28 days of immunity and 84 days bovine serums carried out serial dilution and heat inactivation complement, mixes with the Asia I type FMDV that virulence is arranged of an amount of (100ID50), carry out the neutralization reaction of 1h after, inject in the suckling mouse body observation 72h (table 2).The result shows: produced high-caliberly at the specific IgG antibody of Asia I type FMDV in the serum of A7 albumen+206 oil+RW03 immune cattle, its antibody horizontal is significantly higher than A7 albumen+206 oil group and inactivated vaccine groups (P＜0.01).
The bovine serum of table 2 immunity is to the provide protection of the suckling mouse of FMDV attack
The RW03 sequence is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1).
(4) the ox body is attacked the poison experiment
For detecting behind the immune cattle under the condition that homotype Asia I type FMDV attacks provide protection, in immunity back 84 days, ox has been carried out challenge test to ox.Merit toxic agent amount is the 104ID50/0.2ml/ head.Observed 10 days, observe and the detail record incidence every day.The result shows: produced high-caliberly at the specific IgG antibody of Asia I type FMDV in the serum of A7 albumen+206 oil+RW03 immune cattle, the protection ratio reaches 80%, is higher than inactivated vaccine group 50% (table 3).From period of disease, PBS control group, A7 albumen+206 oil groups and inactivated vaccine group disease time are at virus attack 3-5 days (Fig. 8), and a head of cattle of A7 albumen+206 oil+RW03 group morbidity was fallen ill at the 10th day, and focus is only at gum, it is later to fall ill, and the state of an illness is lighter.
The resistant function that table 3 immune cattle is attacked FMDV
Annotate :+expression blister-expression occurs and blister do not occur
The RW03 sequence is: 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1).
Sequence table
＜110〉Zheng Weizhong
＜120〉as the deoxy-oligonucleotide of ox and pig immunomodulator and vaccine adjuvant
<160>3
<210>1
<211>27
<212>DNA
<213>artificial
<400>1
tcgcgaacgt?tcgcccgatc?gtcggta 27
<210>2
<211>24
<212>DNA
<213>artificial
<400>2
tcgtcgtttt?gtcgttttgt?cgtt 24
<210>3
<211>20
<212>DNA
<213>artificial
<400>3
tccatgacgt?tcctgacgtt 20
Claims (17)
1. the strand deoxy-oligonucleotide of a class synthetic, its sequence comprise 5 '-tcgcgaacgttcgcccgatcgtcggta-3 ' (SEQ ID NO1), can stimulate the mammalian immune proliferation of cells, have the effect of immunoregulation effect and animal vaccine adjuvant.
2. strand deoxy-oligonucleotide as claimed in claim 1, their phosphodiester bond can be non-sulfurized, also can be part or full sulfurized, also can be through chemically modified, and its base can be replaced by rare base.
3. the single chain deoxynucleotide that is the synthetic of core texture as each described single chain deoxynucleotide among the claim 1-2.
The change that is core texture as each described single chain deoxynucleotide among the claim 1-2 one to the single chain deoxynucleotide of several bases.
5. be the interpolation of core texture as each described single chain deoxynucleotide among the claim 1-2 or deleted a single chain deoxynucleotide to several bases that base is added or the position of deletion can occur in the inside or the two ends of this core texture.
6. as each described single chain deoxynucleotide among the claim 1-5, they can stimulate the hyperplasia of ox immunocyte, have immunoregulation effect
7. as each described single chain deoxynucleotide among the claim 1-5, they can stimulate pig immunocyte proliferation of cells, have immunoregulation effect.
8. as each described single chain deoxynucleotide among the claim 1-5, they can obviously strengthen the generation of ox foot and mouth disease recombinant protein vaccine specific antibody.
9. as each described single chain deoxynucleotide among the claim 1-5, they can obviously strengthen the generation of Schweineseuche recombinant protein vaccine specific antibody.
10. as each described single chain deoxynucleotide among the claim 6-9, they can be used as and strengthen the adjuvant of ox with foot and mouth disease recombinant protein vaccine immune effect.
11. as each described single chain deoxynucleotide among the claim 6-9, they can be used as and strengthen the adjuvant of pig with foot and mouth disease recombinant protein vaccine immune effect.
12. as each described single chain deoxynucleotide among the claim 6-9, they can be used as and strengthen the adjuvant of ox with foot and mouth disease recombinant protein vaccine immune effect.
13. as each described single chain deoxynucleotide among the claim 1-5, they can be used as and strengthen the adjuvant of pig with immune effect of vaccine, described pig comprises attenuated vaccine, inactivated vaccine, polypeptide vaccine, subunit vaccine and recombinant protein vaccine with vaccine.
14. as each described single chain deoxynucleotide among the claim 1-5, they can be used as and strengthen the adjuvant of ox with immune effect of vaccine, described ox comprises attenuated vaccine, inactivated vaccine, polypeptide vaccine, subunit vaccine and recombinant protein vaccine with vaccine.
15. as each described single chain deoxynucleotide in the claim 13, they can use the vaccine adjuvant combined utilization with other pig.
16. as each described single chain deoxynucleotide in the claim 14, they can use the vaccine adjuvant combined utilization with other ox.
17. as each described single chain deoxynucleotide among the claim 1-5, they can be used as the adjuvant that strengthens the vaccine immunity of animals effect, described animal vaccine comprises attenuated vaccine, inactivated vaccine, polypeptide vaccine, subunit vaccine and recombinant protein vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101381623A CN102212524A (en) | 2010-04-02 | 2010-04-02 | Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101381623A CN102212524A (en) | 2010-04-02 | 2010-04-02 | Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102212524A true CN102212524A (en) | 2011-10-12 |
Family
ID=44744061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101381623A Withdrawn CN102212524A (en) | 2010-04-02 | 2010-04-02 | Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102212524A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813921A (en) * | 2012-08-16 | 2012-12-12 | 中国农业科学院兰州兽医研究所 | Novel compound aluminum hydroxide adjuvant for foot-and-mouth disease vaccine and vaccine preparation method |
| CN105420243A (en) * | 2015-12-09 | 2016-03-23 | 中国农业科学院特产研究所 | Synthetic mink special CpG ODN and application thereof |
| CN110004150A (en) * | 2018-08-01 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity |
| CN110420323A (en) * | 2019-08-07 | 2019-11-08 | 山东省农业科学院奶牛研究中心 | A kind of Mycoplasma bovis inactivated vaccine and its preparation method and application |
-
2010
- 2010-04-02 CN CN2010101381623A patent/CN102212524A/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813921A (en) * | 2012-08-16 | 2012-12-12 | 中国农业科学院兰州兽医研究所 | Novel compound aluminum hydroxide adjuvant for foot-and-mouth disease vaccine and vaccine preparation method |
| CN102813921B (en) * | 2012-08-16 | 2014-04-16 | 中国农业科学院兰州兽医研究所 | Novel compound aluminum hydroxide adjuvant for foot-and-mouth disease vaccine and vaccine preparation method |
| CN105420243A (en) * | 2015-12-09 | 2016-03-23 | 中国农业科学院特产研究所 | Synthetic mink special CpG ODN and application thereof |
| CN105420243B (en) * | 2015-12-09 | 2018-02-13 | 中国农业科学院特产研究所 | The specific CpG ODN of artificial synthesized mink and its application |
| CN110004150A (en) * | 2018-08-01 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity |
| CN110004150B (en) * | 2018-08-01 | 2023-03-10 | 中国农业科学院兰州兽医研究所 | CpG oligonucleotide sequence with immune enhancement activity and application thereof |
| CN110420323A (en) * | 2019-08-07 | 2019-11-08 | 山东省农业科学院奶牛研究中心 | A kind of Mycoplasma bovis inactivated vaccine and its preparation method and application |
| CN110420323B (en) * | 2019-08-07 | 2023-02-03 | 山东省农业科学院奶牛研究中心 | Mycoplasma bovis inactivated vaccine and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107012149A (en) | Regulate and control immunomodulatory oligonucleotide (IRO) compound of the immune response based on TOLL sample acceptors | |
| CN101821412A (en) | TOLL sample receptor modulators | |
| CN101287742A (en) | Immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response | |
| ES2624767T3 (en) | Enhancement of autoimmune and inflammatory disease treatments using immunoregulatory oligonucleotides (IRO) antagonists of TLR7 and TLR9 | |
| CN101795715A (en) | Stabilized immune modulatory RNA (SIMRA) compounds | |
| CN101918544A (en) | Improve immunoreactive method | |
| CN108330108A (en) | CH-GD-12-2014 plants of 3 type inactivated vaccine of Ana 1 aviadenovirus | |
| EP3456350A1 (en) | Aluminum hydroxide gel-sodium chloride composite immunologic adjuvant, and preparation method therefor and application thereof | |
| WO2006053861A1 (en) | Oligonucleotides that induce the secretion of gm-csf | |
| CN102212524A (en) | Oligodeoxyribonucleotide used as immunomodulator of cattle and pig and vaccine adjuvant | |
| CN110004150B (en) | CpG oligonucleotide sequence with immune enhancement activity and application thereof | |
| EP4130268A1 (en) | Cpg odn having immunoregulatory function and use thereof | |
| WO2010081257A1 (en) | Composition comprising bacillus calmette guerin polysaccharides and bacillus calmette guerin nucleic acids and use of preparing medicament thereof | |
| CN100497370C (en) | Bursopoietin extracting method and its use in disease treating and immune | |
| CN101732704B (en) | Method for preparing inactivated bivalent vaccine against rabbit hemorrhagic disease and pasteurella multocida disease | |
| CN109797139A (en) | 3 type duck hepatitis A virus low virulent strain CH-P60 of one kind and its application | |
| CN107174657A (en) | Prepare the method for the antigen composition of targeting glioma and glioma stem cells and the vaccine containing the antigen composition | |
| CN101701218A (en) | Immune regulatory oligodeoxynucleotide for inhibiting differentiation of Th1 and Th17 and application thereof | |
| CN109234280B (en) | Sika deer specific CpG oligodeoxynucleotide and application thereof | |
| CN103773769B (en) | Adorned oligodeoxynucleotide molecule containing CpG sequence units and application thereof | |
| CN102218139A (en) | Medicament for treating and/or preventing viral infection | |
| CN102813920A (en) | Vaccine adjuvant | |
| CN106256905A (en) | A kind of CpG ODN sequence that Ctenopharyngodon idellus is had immune-enhancing activity and application thereof | |
| CN107149671B (en) | Pharmaceutical composition and application thereof | |
| Wang et al. | Screening of novel immunostimulatory CpG ODNs and their anti-leukemic effects as immunoadjuvants of tumor vaccines in murine acute lymphoblastic leukemia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C53 | Correction of patent for invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 5, No. 130021, No. 1, No. 1, street, No. 661, Silicon Valley street, Changchun hi tech Development Zone, Jilin Applicant after: Yu Zhan Address before: 100017, Beijing, Chaoyang District Beiyuan green home media village 1603 days garden building, room 6 Applicant before: Yu Zhan |
|
| C04 | Withdrawal of patent application after publication (patent law 2001) | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20111012 |