CN100439386C - Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine - Google Patents

Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine Download PDF

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CN100439386C
CN100439386C CNB031198414A CN03119841A CN100439386C CN 100439386 C CN100439386 C CN 100439386C CN B031198414 A CNB031198414 A CN B031198414A CN 03119841 A CN03119841 A CN 03119841A CN 100439386 C CN100439386 C CN 100439386C
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vaccine
modification
thio
oligonucleotide
dna
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CN1526719A (en
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王丽颖
包木胜
于永利
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Huapu Biotechnology Hebei Co ltd
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Changchun Huapu Biotechnology Co Ltd
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Abstract

The present invention provides several kinds of single-strand deoxyoligonucleotides containing CpG, particularly single-strand deoxyoligonucleotides containing CpG capable of reinforcing the immune effects of hydrophobia vaccines, hepatitis B vaccines, influenza virus vaccines and other protein vaccines. The biological preparations of the single-strand deoxyoligonucleotides containing CpG, the hydrophobia vaccines, the hepatitis B vaccines, the influenza virus vaccines and the protein vaccines, and the single-strand deoxyoligonucleotides containing CpG are used for preparing biological preparations which are capable of reinforcing the immunological effects of the hydrophobia vaccines, the hepatitis B vaccines, the influenza virus vaccines and the other protein vaccines.

Description

What strengthen the protein immune effect of vaccine contains CpG strand deoxy-oligonucleotide
Invention field
The present invention relates to several and contain CpG strand deoxy-oligonucleotide, particularly relate to can strengthen rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein immune effect of vaccine contain CpG strand deoxy-oligonucleotide; And these contain the purposes that CpG strand deoxy-oligonucleotide is used to prepare the biotechnological formulation that can strengthen rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein immune effect of vaccine.
Background of invention
Before more than 100 year, first has carried out the experiment of using bacterial extract treatment bacterial infection disease to Coley in the world, and he reaches a conclusion: the extract of bacterium is a kind of preparation that can be used for the treatment of communicable disease.
1984, Tokunaga T etc. found can suppress from the DNA that bacille Calmette-Guerin vaccine (BCG) extracts the growth of multiple animal tumor, had the human peripheral blood single nucleus cell of activation and mouse spleen NK cell (NK) cell activity, the generation of inducing interferon (IFN).Non-vertebrate DNAs has above-mentioned functions, the DNA of vertebrates and plant is quite different, these functions depend on CpG structure (Tokunaga T in the dna molecular, Antitumor activity of deoxyribonucleic acid fractionfrom Mycobacterium bovis BCG.I.Isolation, physicochemical characterization, and antitumor activity, JNCI, 72:955.1984).
CpG is the dinucleotides that is connected into by phosphoric acid by cytosine(Cyt) and guanine.C represents cytosine(Cyt), and G represents guanine, and p represents phosphoric acid, and cytosine(Cyt) is positioned at 5 ' end.The bacterium of the multiple CpG of having structure and viral DNA are dangerous signal to vertebrate immunity system, can activate the panimmunity cell and start bacterium and viral resistance mechanisms.Studies show that in recent years, the oligonucleotide single stranded DNA that contains one or more CpG (CpG ODN) of synthetic also can show potent immunostimulant and immunoregulation effect.CpG ODN can the powerful function that strengthens B cell, T cell, NK cell, antigen presenting cell (monocyte, scavenger cell and dendritic cell) and neutrophil leucocyte, show tangible potential applicability in clinical practice (Weiner GJ, The immunobiology andclinical potential of immunostimulatory CpG oligodeoxynucleotides.J LeukocBiol 2000Oct; 68 (4): 455-63).
Because sequence, the especially difference of the sequence of CpG both sides, CpG ODN can have diversified form, the immunostimulant and the immunoregulation effect of performance different properties, varying strength.CpG ODN can show the kind dependency, promptly a kind of animal is showed the CpGODN of immune enhancing function, then may not show immune enhancing function (Kamstrup S same or equivalence another kind of animal or human, et al.Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides, Vet Microbiol 2001 Feb 26; 78 (4) 352-62; Gunther Hartmann, et al.Delineation of a CpG PhosphorothioateOligodeoxynucleotide for Activating Primate Immune Responses In Vitro andIn Vivol The Journal of Immunology, 2000,164:1617-1624).
Studies show that, some CpG ODN has potent immuno-potentiation, can strengthen the function of B cell, T cell, NK cell, antigen presenting cell (monocyte, scavenger cell and dendritic cell) and neutrophil leucocyte, show tangible clinical value (Weiner GJ, Theimmunobiology and clinical potential of immunostimulatory CpGoligodeoxynucleotides.J Leukoc Biol 2000 Oct; 68 (4): 455-63).Proofs such as Cynthia L., CpG ODN can obviously strengthen immune effect (the Cynthia L.et al.CpG DNA can induce strong Th1 humoral and cell-mediatedimmune responses against hepatitis B surface antigen in young mice.Proc.Natl.Acad.Sci.USA.Vol.95 of reorganization hepatitis B surface antigen(HBsAg) (HBsAg), 26,15553-15558, December 22,1998).Compare with the independent immunity of HBsAg, the immune together BALB/c mouse of HBsAg and CpG ODN can make the titre of its anti-HBsAg antibody increase 5-to 40-times of (Gunther Hartmann, et al.Delineation of a CpGPhosphorothioate Oligodeoxynucleotide for Activating Primate ImmuneResponses In Vitro and In Vivol The Journal of Immunology, 2000,164:1617-1624).
In addition, CpG ODN can obviously strengthen rabies vaccine, the hemophilus influenzae vaccine, diphtheria-Whooping cough-tetanus vaccine and other protein vaccine such as respiratory syncytial virus (RSV) vaccines, foot and mouth disease virus (foot-and-mouth disease virus) vaccine, the immune effect of immunodeficiency virus vaccine many vaccines such as (HIV gp120), CpG ODN can obviously strengthen mononuclearcell hyperplasia and expression CD19 in the peripheral blood, promote the expression of TNF and IL-6.
Summary of the invention
Summary of the invention
One of purpose of the present invention provides several and contains CpG strand deoxy-oligonucleotide, and what particularly can strengthen rabies vaccine, hepatitis B vaccine, influenza vaccines and other protein immune effect of vaccine contains CpG strand deoxy-oligonucleotide.They are made of the oligonucleotide single strand dna that contains one or more CpG, and its phosphodiester bond can be partial vulcanization, and all sulfurized also can be unvulcanized.
Preferably, the CpG of containing strand deoxy-oligonucleotide of the present invention has the sequence shown in the SEQ ID NO:1-81.
Two of purpose of the present invention provides the biotechnological formulation that contains the CpG of containing strand deoxy-oligonucleotide of the present invention and rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein vaccine.Compare to common rabies vaccine, hepatitis B vaccine, influenza vaccines and other protein vaccine, the immune effect of these biotechnological formulations is significantly improved.
Three of purpose of the present invention provides the purposes that the CpG of containing strand deoxy-oligonucleotide of the present invention is used to prepare the biotechnological formulation that can strengthen rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein immune effect of vaccine.
In addition, it is pointed out that aspect and creationary beneficial effect that other has substantive distinguishing features of the present invention can directly be known by inference to those skilled in the art on the basis of the application's contextual disclosure.
Brief Description Of Drawings
Fig. 1 has shown that CpG stimulates peripheral blood mononuclear cell (48h) to produce the experimental result of CD19.
Fig. 2 has shown that CpG stimulates peripheral blood mononuclear cell to produce the experimental result of IL-6.
Embodiment
In the context of the present invention, employed term generally has the implication of those of ordinary skill in the art's common sense unless otherwise indicated.
Of the present invention several contain CpG strand deoxy-oligonucleotide, the CpG strand deoxy-oligonucleotide that contains that particularly can strengthen rabies vaccine, hepatitis B vaccine, influenza vaccines and other protein immune effect of vaccine is made of the oligonucleotide single strand dna that contains one or more CpG, its phosphodiester bond can be partial vulcanization, all sulfurized also can be unvulcanized.Preferably, the CpG of containing strand deoxy-oligonucleotide of the present invention has sequence as follows:
101 5’-TcgTCgAgggCgCCggTgAC-3’
102 5’-TCgTCgCCggTgggggTgTg-3’
103 5’-TCgTCgTACgCAATTgTCTT-3’
104 5’-TCgCCTCgTCgCCTTCgAgC-3’
201 5’-TCgCCCACCggTgggggggg-3’
202 5’-TCgTCgCAgACCggTCTgggg-3’
203 5’-gggggACgTCgCCggggggg-3’
204 5’-ggATCCgTACgCATgggggg-3’
205 5’-TCgTCgCggCCggCgCCCCC-3’
206 5’-TCgTCgCggCCgCgAggggg-3’
301 5’-TCgTCgTTACCgATgACgTCgCCgT-3’
302 5’-TCgTCgggTgCgACgTCgCAgggggg-3’
303 5’-TCgTCgggTgCgACgATCgTCgggggg-3’
304 5?’-TCgTCgTTTgCATCgATgCAgTCgTCgTT-3’
305 5’-TCgTCgTTTgCATCgATgCAggggggg-3’
306 5’-ACCggTATCgATgCCggTgggggg-3’
307 5’-ggggTCCATgACgTTCCTgAAgggggg-3’
308 5’-TCgTCgTTTTgACgATCgTCgggggg-3’
309 5’-TTCgTCgTTTgATCgATgTTCgTTgggggg-3’
310 5’-TTCgTCgTTgTgATCgATgggggg-3’
311 5’-TATCgATgTTTTCgTCgTCgTTgggggg-3’
312 5’-TTCgTTgCATCgATgCATCgTTgggggg-3’
313 5’-TTCgCTTCgCTTTTCgCTTCgCTT-3’
601 5’-TCgAggACAAgATTCTCgTgC-3’
602 5’-TCgAggACAAgATTCTCgTgCAggCC-3’
603 5’-TCgTgCAggCCAACgAggCCg-3’
604 5’-ACCgCCAAggAgAAgCCgCAggAggg-3’
605 5’-TCgTTgCCgTCggCCC-3’
606 5’-TACAACggCgAggAATACC-3’
607 5’-TCggCACgCgACgTgCTggCCgTCgTTTCC-3’
608 5’-gTACAACggCgAggAATACCT-3’
609 5’-ACCgTCgTTgCCgTCggCCC-3’
610 5’-TgCTggCCgTCgTT-3’
611 5’-gTCggCACgCgACg-3’
612 5’-gTCggCACgCgACgggggg-3’
613 5’-gTCggCACgCgACgCCCCCC-3’
614 5’-TCgTTgCCgTCggCCCCCCCCC-3’
615 5’-TCgTTgCCgTCggCCCCCC-3’
616 5’-TCgTTgCCgTCggCCCCC-3’
617 5’-TCgTTgCCgTCggCCCC-3’
618 5’-TCgTTgCCgTCggCCCCCCC-3’
619 5’-TCgTTgCCgTCgg-3’
620 5’-TCgTTgCCgTCggg-3’
621 5’-TCgTTgCCgTCgggg-3’
622 5’-TCgTTgCCgTCggggg-3’
623 5’-TCgTTgCCgTCgggggg-3’
624 5’-TCgTTgCCgTCggggggg-3’
625 5’-TCgTTgCCgTCgggggggg-3’
626 5’-TCgTTgCCgTCggggggggg-3’
627 5’-TCgAggACAAgATTCTCgT-3’
628 5’-TCCCgCTggACgTT-3’
629 5’-TCggCACgCgACgTgCTggCCgTCgTT-3’
631 5’-TCgTCgCgCCgTCACgggggg-3’
632 5’-TCgTgTgCgTgCCgTTggg-3’
633 5’-TCgTCgCCgTTgggCggg-3’
634 5’-TCgTCgACgTCgTTgggCggg-3’
635 5’-TCgCAgTTgTCgTAACgTTgggCggg-3’
636 5’-TTACCggTTAACgTTggCCggCC-3’
637 5’-ACCggTTAACgTTgTCCCCgggg-3’
638 5’-TCgTCgTTggTATgTT-3’
639 5’-TCgTCgTCgTCgTTgTCgTT-3’
640 5’-TCgTCgTCgTCgTTgTCgTTgggg-3’
641 5’-TCgTTCggggTgCCg-3’
642 5’-TCgTTCggggTAACgATT-3’
643 5’-TCgTTCggggTAACgTT-3’
644 5’-TCgTTCggggTACCgAT-3’
645 5’-TCgTTCggggTACCgATgggg-3’
646 5’-TCgTTgCgCTCCCATgCCgggggg-3’
647 5’-TCgTCgTTTCgTCgTTgggg-3’
648 5’-TCgTTgTCgTTTCgCTgCCggCggggg-3’
649 5’-CgTTgACgATCgTCCCATggCggg-3’
650 5’-TCTgCggCCTTCgTCg-3’
651 5’-TAgTAACCggTCCggCgCCCCC-3’
652 5’-TTgCAgCgCTgCCggTggg-3’
653 5’-TCgTACggCCgCCgTACggCggg-3’
654 5’-CggCCCATCgAgggCgACggC-3’
655 5?’-TCgCgTCgACTCCCCTCgAgggg-3’
656 5’-TCgTCgTCgACTCgTggTCggggg-3’
657 5’-TCgggCgCCCgATCgggggg-3’
658 5’-TCgTCggTCTTTCgAAATT-3’
659 5’-TCgTgACgTCCTCgAgTT-3’
Above-described sequence can be partial vulcanization, and all sulfurized also can be unvulcanized.
CpG single chain deoxynucleotide of the present invention can for example adopt the solid phase phosphoramidite triester method to produce by known method production.Following embodiment has at length exemplified a kind of method of producing CpG single chain deoxynucleotide of the present invention.
Contain the purposes that CpG strand deoxy-oligonucleotide is used for preparing the biotechnological formulation that can strengthen rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein immune effect of vaccine at these, the ratio that contains the usage quantity of CpG strand deoxy-oligonucleotide and rabies vaccine, hepatitis B vaccine, influenza virus vaccine and other protein vaccine is 1: 1-100: 1 (mol ratio).
The use-pattern of the CpG of containing strand deoxy-oligonucleotide of the present invention comprises with conventional protein vaccine, dna vaccination mixes use, strengthens immune effect of vaccine, uses by the linking agent covalent coupling with conventional protein vaccine with adjuvant (as aluminium adjuvant, the freund's adjuvant etc.) combined utilization of using always;
Immunization ways comprises the immunization ways that subcutaneous application, mucomembranous surface application, muscle application, gastrointestinal applications, abdominal cavity application etc. are commonly used.
CpG strand deoxy-oligonucleotide is used before can be the conventional vaccine immunity duration of service, is used simultaneously with conventional vaccine, use after the conventional vaccine immunity.
The usage quantity of CpG strand deoxy-oligonucleotide is 1-500 microgram/mouse in mouse test, as carries out human trial and then convert by the conversion method of standard.
Below in conjunction with concrete preparation embodiment and biology effect embodiment, and the present invention is described in further detail with reference to accompanying drawing.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
Embodiment
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art, and for example synthetic employing solid phase phosphoramidite triester method, ELISA adopt indirect method.
In following embodiment, the source of agents useful for same, trade(brand)name and/or be necessary to list its moiety person are all only indicated once.Do not giving unnecessary details foregoing if no special instructions at used identical reagent thereafter.
The preparation of embodiment 1CpG single chain deoxynucleotide (it is synthetic that the worker is given birth in Shanghai)
The synthetic solid phase phosphoramidite triester method that adopts, synthetic method (Shanghai is given birth to the worker and provided) is as follows: material and method:
Trichoroacetic acid(TCA) (Trichloroacetic Acid, TCA), controlled pore glass (Controlled PoreGlass), DMT (dimethoxytrityl), tetrazole activator, diacetyl oxide, N-Methylimidazole, ABI dna synthesizer, high performance liquid chromatography chromatograph etc.
A, T, four kinds of nucleotide monomers of C, G: synthetic used monomer is a nucleoside phosphoramidites, is the Nucleotide through chemically modified, contains following several functional group:
1.3 ' the last diisopropylamino of position P, the functional group that condensation is used
2.3 ' the last nitrile ethyl of position P, protecting group is sloughed behind synthetic the finishing.
3.5 '-Dmt, protecting group is sloughed before the condensation.
4.A and the phenylformic acid protecting group on the heterocyclic amino group of C, slough behind synthetic the finishing.
5.G the different propionyl protecting group on the last purine skeleton amino is sloughed behind synthetic the finishing.
Concrete reactions steps is as follows:
One, deprotection base
With trichoroacetic acid(TCA) (Trichloroacetic Acid; TCA) slough the blocking group dimethoxytrityl (DMT) that is attached at the Nucleotide on the controlled pore glass (Controlled Pore Glass); obtain free 5 '-hydroxyl terminal, for next step condensation reaction.
Two, activation
With the nucleotide monomer and the tetrazole activator mix of phosphoramidite protection and enter synthetic post; (its 3 '-end is activated to form phosphoramidite tetrazolium active intermediate; but 5 '-end is protected by DMT still), this intermediate will with the Nucleotide generation condensation reaction of the base of deprotection on the controlled pore glass.
Three, connect
When phosphoramidite tetrazolium active intermediate runs on the controlled pore glass the Nucleotide of deprotection base, will with its 5 '-hydroxyl generation affinity reaction, condensation is also sloughed tetrazolium, this moment, the synthetic oligonucleotide chain prolonged a base forward.
Four, sealing
Be extended in circulating reaction subsequently for 5 ' of the reaction-hydroxyl that has neither part nor lot in that prevents to be connected on the controlled pore glass after the condensation reaction; often seal this terminal hydroxy group by acetylize, general acetylation reagent mixes formation with diacetyl oxide and N-Methylimidazole etc.
Five, oxidation
Nucleotide monomer is to be connected with oligonucleotide on being connected in controlled pore glass by inferior phosphide key during condensation reaction, and inferior phosphide key instability, easily by acid, basic hydrolysis, this moment, the tetrahydrofuran solution of iodine commonly used was converted into phosphotriester with inferior phosphinylidyne, obtained stable oligonucleotide.
Through after above five steps; a deoxynucleotide is just linked on the Nucleotide of controlled pore glass; after sloughing blocking group DMT on the deoxynucleotide 5 '-hydroxyl that newly connects with trichoroacetic acid(TCA) equally again, repeat above activation, connection, sealing, oxidising process and can obtain a dna fragmentation crude product.At last to its cut, the deprotection base (generally adopts the benzoyl protection to A, C base; The G base is protected with isobutyryl; The T base needn't be protected; Phosphorous acid is with nitrile ethyl protection), purifying (commonly used have HAP, PAGE, HPLC, C18, methods such as OPC), synthetic aftertreatment such as quantitative can obtain meeting the oligonucleotide fragment of requirement of experiment.
Unvulcanized CpG single chain deoxynucleotide synthetic on ABI 3900 dna synthesizers (worker's biotechnology Services Co., Ltd is given birth in Shanghai), and the synthetic employing substitution method of sulfuration and the CpG single chain deoxynucleotide of partial vulcanization entirely, synthetic on ABI 394DNA synthesizer (worker's biotechnology Services Co., Ltd is given birth in Shanghai).
The experiment of embodiment 2CpG stimulation human peripheral blood mononuclearcell hyperplasia
(1) separation of human peripheral blood single nucleus cell
1, reagent and material:
People's whole blood of anticoagulant heparin: Central Blood Ban, Changchun City.
Ficoll-Hypaque: proportion 1.077 ± 0.001, Beijing ancient cooking vessel state Bioisystech Co., Ltd.The plant and instrument that cell cultures is commonly used: the suction pipe of cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, water distillation apparatus, vacuum pump, Tissue Culture Flask, bacterial filter, filtration bottle, all size, sample injector, dropper, blood counting chamber, horizontal whizzer etc.
The RPMI1640 nutrient solution:
RPMI1640 (GIBCOBRL) 10.4 grams that contain L-glutaminate
Sodium bicarbonate 2.0 grams
Gentamicin 100,000 units
Add tri-distilled water to 1000 milliliters of volumes
0.22 the degerming of filter membrane vacuum pump suction filtration, the packing of micron.
The calf serum deactivation: calf serum (Invitrogen)-20 ℃ taking-up, after 4 ℃ of refrigerators melt, 56 ℃ of water-bath 30min.
The RPMI1640 of 10% calf serum:
10 milliliters of the calf serums of deactivation
90 milliliters of RPMI RPMI-1640s
The preparation of Hank ' s liquid (no calcium ion, magnesium ion):
Sodium-chlor 8.0 grams
Repone K 0.4 gram
Sodium phosphate dibasic (being with a crystal water) 0.06 gram
Potassium primary phosphate 0.06 gram
Sodium bicarbonate 0.35 gram
Glucose 1.0 grams
Phenol red 0.02 gram
Add distilled water to 1000 milliliter.
To dissolve after the above-listed composition mixing, 8 pounds of 15min sterilizations, 4 ℃ of refrigerators are preserved.Face the time spent and transfer pH to 7.3～7.6 with 7.4%NaHCO3.
The blue staining fluid of 2% tongue phenol:
Blue 2 grams of tongue phenol
100 milliliters in physiological saline
2, method:
It is that the ratio of parting liquid and peripheral blood is about 2: 1 on 1.077 ± 0.001 the Ficoll-Hypaque lymphocyte laminated fluid level that the human peripheral of anticoagulant heparin slowly slowly is added on proportion along tube wall.
Horizontal centrifuge is centrifugal 1, and 000xg 15-20min is divided into 3 layers in the pipe of centrifugal back, draws the narrow band of white cloud and mist layer with suction pipe, inserts in another pipe.
Add to wait doubly or with Hank ' the s liquid (serum-free) of upper volume, 800-1,000xg 10-15min abandons supernatant, adds Hank ' s liquid, twice of washed cell.
After last is centrifugal, abandon supernatant, add substratum 2ml, re-suspended cell.
Get a cell suspension and mix the total cellular score in four big grids of blood counting chamber counting, mononuclearcell concentration (cell count/1 ml cells suspension)=4 big grid inner cell sum/4 * 10 with the blue dye liquor of one 0.2% tongue phenol 4* 2 (extension rates).
(2) 3H-TDR mixes test
1, equipment and reagent
The RPMI1640 nutrient solution:
RPMI1640 (GIBCOBRL) 10.4 grams that contain L-glutaminate
Sodium bicarbonate 2.0 grams
Gentamicin 100,000 units
Add tri-distilled water to 1000 milliliters of volumes
0.22 the degerming of filter membrane vacuum pump suction filtration, the packing of micron.
The calf serum deactivation: calf serum (Invitrogen)-20 ℃ taking-up, after 4 ℃ of refrigerators melt, 56 ℃ of water-bath 30min.
The RPMI1640 of 10% calf serum:
10 milliliters of the calf serums of deactivation
90 milliliters of RPMI RPMI-1640s
(HCl regulates pH to 7.0 to TE damping fluid TE damping fluid for 10mM Tris, 1mM EDTA.Autoclaving, membrane filtration) the CpG ODN of preparation
3H-TDR (contains 25 μ Ci among the 25 μ l 3H-TDR)
96 orifice plates (flat)
Eppendorf manages (0.5ml, 1.5ml)
Cell harvestor
The liquid flashing counting device
37 ℃ of CO 2Incubator
2, method
With perfect medium mediator peripheral blood mononuclear cell (PBMC) to final concentration 3 * 10 6Individual/ml.
PBMC is added in 96 orifice plates every hole 100 μ l (3 * 10 5Individual/hole), wherein contain the CpG that final concentration is the sulfo-of 0.6 μ g/ml, each CpG three multiple hole.
Cultivate after 24-48 hour, after the adding dilution of every hole 3H-TDR ( 3H-TDR is diluted to 25Ci/ml with perfect medium) 20 μ l, promptly every hole includes 0.5 μ Ci 3H-TDR continues to cultivate 16-18d.
To filter paper, 60 ℃ of incubator baking filter paper 2-3h put into liquid with filter paper and dodge bottle, add scintillation solution, survey the cpm value with the cell harvestor collecting cell
(3) experimental result (cpm value):
The cpm value in three multiple holes of negative control (not adding CpG ODN): 574 544 390
Below be the cpm value in three multiple holes of each CpG ODN stimulating group:
203：826 1450 1302
205：12514 10594 11006
302：7252 6630 6248
303：5284 5624 5396
305：2950 9136 10120
306：6770 3830 3844
307：1740 1718 1962
304：11524 13148 11574
310：10854 10210 10258
607：12406 15204 14110
608：3118 2446 2994
610：4228 4362 4624
619：2196 7738 7304
623：8598 8056 7576
631：6152 6688 6240
634：12038 12068 12478
639：4194 17894 17652
640：16078 16966 17026
645：12100 12424 11556
647：17022 14588 16372
654：5246 4272 5010
656：11704 12446 12378
657：4244 3910 4200
658：16206 16602 15506
659：4332 16392 16284
Embodiment 3CpG ODN stimulates people's periphery mononuclearcell to express CD19
(1) separation of human peripheral blood single nucleus cell
1, reagent and material:
People's whole blood of anticoagulant heparin: Central Blood Ban, Changchun City.
Ficoll-Hypaque: proportion 1.077 ± 0.001, Beijing ancient cooking vessel state Bioisystech Co., Ltd.
The plant and instrument that cell cultures is commonly used: the suction pipe of cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, water distillation apparatus, vacuum pump, Tissue Culture Flask, bacterial filter, filtration bottle, all size, sample injector, dropper, blood counting chamber, horizontal whizzer etc.
The RPMI1640 nutrient solution:
RPMI1640 (GIBCOBRL) 10.4 grams that contain L-glutaminate
Sodium bicarbonate 2.0 grams
Gentamicin 100,000 units
Add tri-distilled water to 1000 milliliters of volumes
0.22 the degerming of filter membrane vacuum pump suction filtration, the packing of micron.
The calf serum deactivation: calf serum (Invitrogen)-20 ℃ taking-up, after 4 ℃ of refrigerators melt, 56 ℃ of water-bath 30min.
The RPMI1640 of 10% calf serum:
10 milliliters of the calf serums of deactivation
90 milliliters of RPMI RPMI-1640s
The preparation of Hank ' s liquid (no calcium ion, magnesium ion):
Sodium-chlor 8.0 grams
Repone K 0.4 gram
Sodium phosphate dibasic (being with a crystal water) 0.06 gram
Potassium primary phosphate 0.06 gram
Sodium bicarbonate 0.35 gram
Glucose 1.0 grams
Phenol red 0.02 gram
Add distilled water to 1000 milliliter.
To dissolve after the above-listed composition mixing, 8 pounds of 15min sterilizations, 4 ℃ of refrigerators are preserved.Face the time spent and transfer pH to 7.3～7.6 with 7.4%NaHCO3.
The blue staining fluid of 2% tongue phenol:
Blue 2 grams of tongue phenol
100 milliliters in physiological saline
2, method:
It is that the ratio of parting liquid and peripheral blood is about 2: 1 on 1.077 ± 0.001 the Ficoll-Hypaque lymphocyte laminated fluid level that the human peripheral of anticoagulant heparin slowly slowly is added on proportion along tube wall.
Horizontal centrifuge is centrifugal 1, is divided into three layers in the pipe of the centrifugal back of 000xg 15-20min, draws the narrow band of white cloud and mist layer with suction pipe, inserts in another pipe.
Add to wait doubly or with Hank ' the s liquid (serum-free) of upper volume, 800-1,000xg 10-15min abandons supernatant, adds Hank ' s liquid, twice of washed cell.After last is centrifugal, abandon supernatant, add substratum 2ml, re-suspended cell.
Get a cell suspension and mix the total cellular score in four big grids of blood counting chamber counting, mononuclearcell concentration (cell count/1 ml cells suspension)=4 big grid inner cell sum/4 * 10 with the blue dye liquor of one 0.2% tongue phenol 4* 2 (extension rates).
3, the CD19 that expresses with cells were tested by flow cytometry human peripheral blood single nucleus cell (PBMC)
1) transfers person peripheral blood mononuclear cell (PBMC) to final concentration 3 * 10 with perfect medium 6/ ml.PBMC is added in 96 orifice plates every hole 100 μ l (3 * 105/hole).
2) every hole adds the CpG ODN of sulfo-, and final concentration is 0.6 μ g/ml, and every kind of CpG ODN establishes three multiple holes.37 ℃ of 5%CO2 cultivate 24-48h.
3) with 4 ℃ of 1260 rev/mins of centrifugal 5min of 96 orifice plates.
4) abandon supernatant, 4 ℃ of 1260 rev/mins of centrifugal 5min wash twice with the PBS damping fluid.
5) abandon supernatant, with 50 microlitre PBS damping fluid re-suspended cells.
6) 96 orifice plate mixings are flicked by the CD19 antibody that adds marked by fluorescein isothiocyanate at 1: 50 in every hole.Lucifuge is hatched 30min on ice.
7) wash twice with 4 ℃ of 1260 rev/mins of centrifugal 5min of PBS damping fluid.
8) with 200 μ lPBS damping fluid re-suspended cells, and move in the testing tube, carry out flow cytometer (FAGS) and detect.The result as shown in Figure 1.
Embodiment 4 stimulates peripheral blood mononuclear cell to produce IL-6
(1) separation of human peripheral blood single nucleus cell
1, reagent and material:
People's whole blood of anticoagulant heparin: Central Blood Ban, Changchun City.
Ficoll-Hypaque: proportion 1.077 ± 0.001, Beijing ancient cooking vessel state Bioisystech Co., Ltd.
The plant and instrument that cell cultures is commonly used: cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inversion are micro-, the suction pipe of liquid nitrogen container water distillation apparatus, vacuum pump, Tissue Culture Flask, bacterial filter, filtration bottle, all size, sample injector, dropper, blood counting chamber, horizontal whizzer etc.
The RPMI1640 nutrient solution:
RPMI1640 (GIBCOBRL) 10.4 grams that contain L-glutaminate
Sodium bicarbonate 2.0 grams
Gentamicin 100,000 units
Add tri-distilled water to 1000 milliliters of volumes
0.22 the degerming of filter membrane vacuum pump suction filtration, the packing of micron.
The calf serum deactivation: calf serum (Invitrogen)-20 ℃ taking-up, after 4 ℃ of refrigerators melt, 56 ℃ of water-bath 30min.
The RPMI1640 of 10% calf serum:
10 milliliters of the calf serums of deactivation
90 milliliters of RPMI RPMI-1640s
The preparation of Hank ' s liquid (no calcium ion, magnesium ion):
Sodium-chlor 8.0 grams
Repone K 0.4 gram
Sodium phosphate dibasic (being with a crystal water) 0.06 gram
Potassium primary phosphate 0.06 gram
Sodium bicarbonate 0.35 gram
Glucose 1.0 grams
Phenol 0.02 gram
Add distilled water to 1000 milliliter.
To dissolve after the above-listed composition mixing, 8 pounds of 15min sterilizations, 4 ℃ of refrigerators are preserved.Facing the time spent uses
7.4%NaHCO3 transfers pH to 7.3～7.6.
The blue staining fluid of 2% tongue phenol:
Blue 2 grams of tongue phenol
100 milliliters in physiological saline
2, method:
It is 1.077 ± 0.001 Ficoll-Hypaque lymph that the human peripheral of anticoagulant heparin slowly slowly is added on proportion along tube wall
On the cell laminated fluid level, the ratio of parting liquid and peripheral blood is about 2: 1.
Horizontal centrifuge is centrifugal 1, and 000xg 15-20min is divided into three layers in the pipe of centrifugal back, draws the narrow band of white cloud and mist layer with suction pipe, inserts in another pipe.
Add to wait doubly or with Hank ' the s liquid (serum-free) of upper volume, 800-1,000xg 10-15min abandons supernatant, adds Hank ' s liquid, twice of washed cell.
After last is centrifugal, abandon supernatant, add substratum 2ml, re-suspended cell.
Get a cell suspension and mix the total cellular score in four big grids of blood counting chamber counting, mononuclearcell concentration (cell count/1 ml cells suspension)=4 big grid inner cell sum/4 * 10 with the blue dye liquor of one 0.2% tongue phenol 4* 2 (extension rates).
3, the mensuration of IL-6
With perfect medium mediator peripheral blood mononuclear cell (PBMC) to final concentration 3 * 10 6Individual/ml.
PBMC is added in 96 orifice plates every hole 100l (3 * 10 5Individual/hole), wherein containing final concentration is the CpG ODN of the sulfo-of 0.6 μ g/ml, each CpG ODN, three multiple holes.Cultivate after 24-48 hour, get supernatant and survey IL-6.
ELISA test kit (Quantikine R ﹠amp; D) step of mensuration IL-6:
(1) gets all ingredients and standard substance ready;
(2) every hole adds 100ul RD1A test diluent;
(3) add IL-6 standard substance or sample (being supernatant) 100 μ l/ holes, put incubated at room 2h;
(4) dry and wash 4 times with damping fluid;
(5) add 200 μ l/ hole enzyme labelled antibodies, put incubated at room 2h;
(6) dry and wash 4 four times with damping fluid;
(7) add 200 μ l/ hole substrates, put room temperature 20min;
(8) add 50 μ l/ hole stop buffers, the 450nm place value of reading, the result is as shown in Figure 2.
Embodiment 5 strengthens rabies vaccine immune effect experiment (virus attack method)
Rabies Vaccine synergism test (NIH method)
(1) experiment material
The preparation attack strain CVS that attacks strain is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute of the Ministry of Health, and breakdown freeze-drying seed culture of viruses is diluted to 10 -2Suspension, 0.03ml passes 2-3 generation continuously with the intracranial inoculation of body weight 11-13g mouse, select 4-5d the mouse of typical rabies to occur when receiving brain, the mouse brain grind added contain notmal horse sera in right amount or calf serum distilled water is made 20% suspension, the centrifugal 10min of 1000r/min does titration of virus with 10 of body weight 18-20g mouse, meets the requirements, the packing tubule ,-60 ℃ of preservations.
The standard rabies vaccine is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute
(2) experimental procedure
Rabies vaccine grouping: 1. rabies vaccine (Changchun Biological Products Institute provides); 2. rabies vaccine+AL (OH) 33. CpG640 (10 micrograms/mouse)+rabies vaccine; 4. CpG647 (10 micrograms/mouse)+rabies vaccine; 5. CpG640 (10 micrograms/mouse)+AL (OH) 3+ rabies vaccine; 6. CpG647 (10 micrograms/mouse)+rabies vaccine+AL (OH) 3
Every group of rabies vaccine get 5 *, 25 *, 125 * three weaker concns.
The standard rabies vaccine: get 25 *, 125 *, 625 * three weaker concns.Select 15 of body weight 12-14g mouse for use, every abdominal cavity inoculation 0.5ml, at interval after the week again immunity once and reserve 40 LD that are used for measuring challenge virus with batch mouse 50
Attack: mouse is immunity back 14d for the first time, and the effectiveness of measuring strain CVS in advance is that every 0.03ml contains 5-100 * LD 50Virus quantity.Viral liquid is pressed 10 0, 10 -1, 10 -2, 10 - 3Dilution.Use each dilution virus (0.03ml) respectively each group mouse to be carried out attacking in the brain every group of 10 mouse then.15 mouse of CpG+ rabies vaccine group.All mouse are observed 14d from attacking, attack that dead mouse does not take statistics in initial 4 days of the back, and mouse dead and that present the typical brain disease symptoms carries out counting statistics behind the 5d.Vaccine potency is measured and is not less than 2.5 international unit (IU)/agent is qualified.
Figure C0311984100211
The results are shown in following table (table 1-table 8).
Table 1 rabies vaccine (Changchun Biological Products Institute) immune effect
ED 50＝1.4+(50-26.1)×0.7/(71.4-26.1)＝1.77
IU＝10 (1.77-2.29)×6.7＝2.02
Table 2 rabies vaccine (Changchun Biological Products Institute)+AL (OH) 3The group immune effect
Figure C0311984100213
ED 50＝1.4+(50-20)×0.7/(57.9-20)＝1.95
IU＝10 (1.95-2.29)×6.7＝3.06
Table 3CpG640+ rabies vaccine (Changchun Biological Products Institute) group immune effect
Figure C0311984100221
ED 50＝1.4+(50-13.6)×0.7/(66.7-13.6)＝1.88
IU＝10 (1.88-2.29)×6.7＝2.60
Table 4CpG647+ rabies vaccine (Changchun Biological Products Institute) group immune effect
Figure C0311984100222
ED 50＝1.4+(50-18.2)×0.7/(68.4-18.2)＝1.84
IU＝10 (1.84-2.29)×6.7＝2.37
Table 5CpG640+ rabies vaccine (Changchun Biological Products Institute)+AL (OH) 3The group immune effect
ED 50＝1.4+(50-13)×0.7/(61.1-13)＝1.94
IU＝10 (1.94-2.29)×6.7＝2.99
Table 6CpG647+ rabies vaccine (Changchun Biological Products Institute)+AL (OH) 3Immune effect
Figure C0311984100232
ED 50＝1.4+(50-17.4)×0.7/(63.2-17.4)＝1.90
IU＝10 (1.90-2.29)×6.7＝2.73
Table 7 standard seedling (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) group immune effect
Figure C0311984100241
ED 50＝2.1+(50-38.1)×0.7/(82.6-38.1)＝2.29
IU＝6.7
Table 8 is attacked malicious measurement result
Figure C0311984100242
(3) conclusion
It is as follows that each organizes the IU value:
Table 1 rabies vaccine group: 2.02
Table 2 rabies vaccine+AL (OH): 3.06
Table 3CpG640+ rabies vaccine group: 2.60
Table 4CpG647+ rabies vaccine group: 2.37
Table 5CpG640+ rabies vaccine+AL (OH): 2.99
Table 6CpG647+ rabies vaccine+AL (OH): 2.73
From each the group result as can be seen, except the 1st group (rabies vaccine group IU=2.02) and the 4th group of (CpG647+ rabies vaccine group IU=2.37) IU＜2.5th, all the other respectively organize IU all greater than 2.5, prove that CpG ODN can strengthen the immune effect of rabies vaccine really.
Each group is tried mortality of mice and is respectively when the extent of dilution of rabies vaccine is 25 times of dilutions:
Table 1 rabies vaccine group: 26.1
Table 2 rabies vaccine+AL (OH): 20
Table 3CpG640+ rabies vaccine group: 13.6
Table 4CpG647+ rabies vaccine group: 18.2
Table 5CpG640+ rabies vaccine+AL (OH): 13
Table 6CpG647+ rabies vaccine+AL (OH): 17.4
The above results shows that CpG640, CpG647 have synergism to rabies vaccine.
Embodiment 6 strengthens rabies vaccine immune effect experiment (antibody testing method)
One, laboratory animal and grouping:
70 7 age in week Kunming mouse, body weight 20 ± 2 gram is divided into 14 groups at random, 5 every group, sub-cage rearing adapts to 2 days earlier before the immunity.
Two, immunization route and dosage:
1-13 organizes mouse: intramuscular injection 1 μ g reorganization rabies vaccine (Changchun Biological Products Institute)/100 μ l PBS adds 10 μ g CpG ODN, divides 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
14 groups: intramuscular injection 1 μ g reorganization reorganization rabies vaccine (Changchun Biological Products Institute)/100 μ lPBS, (no CpG ODN) divides 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
Three, immune programme for children:
In 0, the 21d immunity, immunity back 7d takes a blood sample through caudal artery for the second time, every mouse separates 10ul serum, directly detect antibody titers or put-20 ℃ frozen.
Four, the mensuration of antibody titer (indirect method ELISA)
The reagent equipment:
The bag be cushioned liquid (the 0.05mol/L carbonate buffer solution, pH9.6):
Na 2CO 31.59 gram
NaHCO 32.93 gram
Adding distil water to 1000 milliliter
Lavation buffer solution (PBS-T): contain 0.05% tween 20, pH7.4
K 2HPO 40.2 gram
Na 2HPO 412H 2O 2.9 grams
NaCl 8.0 grams
KCl 0.2 gram
0.5 milliliter of Tween-20
Adding distil water is to 1000ml
Sample diluting liquid:
Bovine serum albumin (BSA) 0.1 gram
Add lavation buffer solution to 100 milliliter
Confining liquid (2%BSA):
Bovine serum albumin (BSA) 2 grams
Add PBS (pH7.4) to 100 milliliters
Substrate buffer solution (phosphoric acid-citrate buffer solution, pH=5.0):
● first liquid (0.1mol/L citric acid)
Citric acid 1.92 grams
Adding distil water to 100 milliliter
● second liquid (0.2mol/L Na 2HPO 4)
Na 2HPO 412H 2O 7.17 grams
Adding distil water to 100 milliliter
Get first liquid 24.3mL, second liquid 25.7mL, adding distil water to 100 milliliter
Tetramethyl biphenyl amine aqueous solution (TMB): 2 mg/ml
10 milligrams of TMB
5 milliliters of ethanol
Substrate solution:
10 milliliters of substrate buffer solutions
0.5 milliliter of TMB (2 mg/ml)
30%H 2O 210 microlitres
Stop buffer:
Dense H 2SO 422ml
Distilled water 178ml
Step
1. bag quilt: being cushioned liquid dilution rabies vaccine (Chinese Changchun Biological Products Institute provides) to the 1-10 mcg/ml with bag, adding enzyme plate,, spends the night in the 100ul/ hole by 4 ℃.
2. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
3. sealing: add the 2%BSA sealing, the 200ul/ hole.37℃，1.5h。
4. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
5. application of sample: test serum is done 400 times of dilutions, 100ul/ hole with sample diluting liquid.The blank hole adds the sample diluent, the 100ul/ hole, and each sample is established 3 multiple holes.37℃，60min。
6. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
7. add enzyme labelled antibody: rabbit anti-mouse igg-HRP (the biological 0.1ml of Beijing ancient cooking vessel state, 1: 1000) is diluted 500 times with lavation buffer solution, 100ul/ hole, 37 ℃, 60min.
8. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
9. colour developing: add substrate solution, the 100ul/ hole.Room temperature lucifuge reaction 10-30min.
10. stop: 50ul/ hole 2M H 2SO 4
11. microplate reader (BIO-RAD Model 550) is surveyed A 450
Five, result
1-13 organizes mouse (intramuscular injection 1 μ g hydrophobia/100 μ l PBS add 10 μ g CpG ODN) 14 groups of mouse (intramuscular injection 1 μ g hydrophobia/100 μ l PBS)
1 group (205): 1) .0.576 0.566 0.587
2).0.682 0.618 0.657
3).0.510 0.538 0.566
4).0.656 0.751 0.715
5).0.658 0.689 0.611
2 groups (302): 1) .0.775 0.734 0.691
2).0.759 0.790 0.719
3).0.630 0.615 0.626
4).0.688 0.657 0.663
5).0.943 0.847 0.899
3 groups (304): 1) .0.824 0.834 0.810
2).0.681 0.658 0.615
3).0.619 0.647 0.656
4).0.607 0.634 0.612
5).0.787 0.768 0.754
4 groups (310): 1) .0.645 0.674 0.652
2).0.749 0.690 0.724
3).0.775 0.784 0.732
4).0.644 0.674 0.646
5).0.688 0.693 0.648
5 groups (607): 1) .0.723 0.719 0.747
2).0.702 0.635 0.697
3).0.818 0.787 0.800
4).0.790 0.786 0.691
5).0.890 0.831 0.817
6 groups (634): 1) .0.754 0.699 0.658
2).0.655 0.606 0.677
3).0.727 0.787 0.719
4).0.693 0.685 0.732
5).0.556 0.588 0.543
7 groups (639): 1) .0.976 0.919 0.923
2).0.768 0.717 0.699
3).0.886 0.876 0.832
4).1.012 1.007 1.188
5).0.868 0.839 0.861
8 groups (640): 1) .0.819 0.836 0.822
2).0.670 0.625 0.611
3).1.283 1.186 1.005
4).1.366 1.212 1.266
5).0.975 0.980 0.931
9 groups (645): 1) .0.876 0.827 0.834
2).0.925 0.898 0.953
3).0.732 0.744 0.712
4).0.850 0.824 0.833
5).0.689 0.723 0.756
10 groups (647): 1) .0.968 0.978 0.951
2).0.983 0.982 1.002
3).0.988 0.980 0.985
4).1.179 1.280 1.214
5).1.006 0.979 0.980
11 groups (656): 1) .0.682 0.672 0.656
2).0.796 0.752 0.743
3).0.658 0.686 0.619
4).0.615 0.597 0.637
5).0.687 0.690 0.711
12 groups (657): 1) .0.778 0.729 0.710
2).0.628 0.710 0.699
3).0.676 0.667 0.701
4).0.635 0.721 0.677
5).0.690 0.682 0.735
13 groups (658): 1) .0.673 0.667 0.712
2).0.615 0.710 0.694
3).0.834 0.876 0.887
4).0.870 0.818 0.823
5).0.673 0.685 0.655
14 groups (659): 1) .1.327 1.289 1.017
2).0.968 0.972 0.991
3).1.117 1.276 0.991
4).0.860 0.874 0.870
5).0.886 0.816 0.854
15 groups (contrast): 1) .0.477 0.423 0.456
2).0.506 0.497 0.486
3).0.399 0.401 0.435
4).0.455 0.427 0.417
5).0.368 0.398 0.406
Background value: 0.332 0.287 0.319
The above results shows, all obvious height of antibody titer and control group in the 1-13 group, and wherein with the 7th group (CpG ODN 639), the 8th group (CpG ODN 640), and the 10th group (CpG ODN 647), the 14th group of (CpG ODN 659) antibody titers raises the most obvious.
Embodiment 7 strengthens the immune effect experiment of hepatitis B vaccine
One. laboratory animal and grouping:
156 7 age in week Kunming mouse, body weight 20 ± 2 gram is divided into 26 groups at random, 6 every group, sub-cage rearing adapts to 2d earlier before the immunity.
Two. immunization route and dosage:
1-25 organizes mouse: intramuscular injection 1 μ g recombinant HBsAg (Chinese Changchun Biological Products Institute provides)/100 μ l PBS adds 10 μ g CpG ODN, divides 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
26 groups: intramuscular injection 1 μ g recombinant HBsAg/100 μ l PBS, do not add CpG ODN, divide 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
Three. immune programme for children:
0d, 21d immunity, back 7 days venous blood collections of immunity for the second time, every mouse separates 10ul serum, directly survey antibody titers or put-20 ℃ frozen.
Four, the mensuration of antibody titer (indirect method ELISA)
The reagent equipment:
The bag be cushioned liquid (the 0.05mol/L carbonate buffer solution, pH9.6):
Na 2CO 31.59 gram
NaHCO 32.93 gram
Adding distil water to 1000 milliliter
Lavation buffer solution (PBS-T): contain 0.05% tween 20, pH7.4
K 2HPO 40.2 gram
Na 2HPO 412H 2O 2.9 grams
NaCl 8.0 grams
KCl 0.2 gram
0.5 milliliter of Tween-20
Adding distil water is to 1000ml
Sample diluting liquid:
Bovine serum albumin (BSA) 0.1 gram
Add lavation buffer solution to 100 milliliter
Confining liquid (2%BSA):
Bovine serum albumin (BSA) 2 grams
Add PBS (pH7.4) to 100 milliliters
Substrate buffer solution (phosphoric acid-citrate buffer solution, pH=5.0):
● first liquid (0.1mol/L citric acid)
Citric acid 1.92 grams
Adding distil water to 100 milliliter
● second liquid (0.2mol/L Na 2HPO 4)
Na 2HPO 412H 2O 7.17 grams
Adding distil water to 100 milliliter
Get first liquid 24.3mL, second liquid 25.7mL, adding distil water to 100 milliliter
Tetramethyl biphenyl amine aqueous solution (TMB): 2 mg/ml
10 milligrams of TMB
5 milliliters of ethanol
Substrate solution:
10 milliliters of substrate buffer solutions
0.5 milliliter of TMB (2 mg/ml)
30%H 2O 210 microlitres
Stop buffer:
Dense H 2SO 422ml
Distilled water 178ml
Step
1. bag quilt: being cushioned liquid dilution HBsAg (Chinese Changchun Biological Products Institute provides) to the 1-10 mcg/ml with bag, adding enzyme plate,, spends the night in the 100ul/ hole by 4 ℃.
2. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
3. sealing: add the 2%BSA sealing, the 200ul/ hole.37℃，1.5h。
4. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
5. application of sample: test serum is done 400 times of dilutions, 100ul/ hole with sample diluting liquid.The blank hole adds the sample diluent, the 100ul/ hole, and each sample is established 3 multiple holes.37℃，60min。
6. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
7. add enzyme labelled antibody: rabbit anti-mouse igg-HRP (the biological 0.1ml of Beijing ancient cooking vessel state, 1: 1000) is diluted 500 times with lavation buffer solution, 100ul/ hole, 37 ℃, 60min.
8. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
9. colour developing: add substrate solution, the 100ul/ hole.Room temperature lucifuge reaction 10-30min.
10. stop: 50ul/ hole 2M H 2SO 4
11. microplate reader (BIO-RAD Model 550) is surveyed A 450
Four, result
1-25 organizes mouse: (intramuscular injection 1 μ g recombinant HBsAg/100 μ l PBS add 10 μ g CpGODN)
1 group (203): 1) .0.562 0.573 0.575
2).0.581 0.573 0.570
3).0.580 0.580 0.576
4).0.561 0.555 0.560
5).0.577 0.578 0.573
6).0.555 0.548 0.559
2 groups (205): 1) .0.762 0.777 0.775
2).0.589 0.582 0.590
3).0.681 0.684 0.686
4).0.600 0.655 0.661
5).0.878 0.885 0.866
6).0.644 0.654 0.668
3 group (302) 1) .0.762 0.777 0.775
2).0.589 0.582 0.590
3).0.681 0.684 0.686
4).0.600 0.655 0.661
5).0.878 0.885 0.866
6).0.644 0.654 0.668
4 group (303) 1) .0.444 0.479 0.500
2).0.498 0.500 0.490
3).0.580 0.584 0.568
4).0.443 0.455 0.462
5).0.478 0.481 0.486
6).0.640 0.645 0.681
5 groups (305): 1) .0.645 0.700 0.665
2).0.749 0.665 0.691
3).0.880 0.883 0.800
4).0.920 0.895 0.914
5).0.900 0.879 0.886
6).0.886 0.876 0.811
6 groups (306): 1) .0.545 0.601 0.577
2).0.557 0.606 0.609
3).0.780 0.893 0.789
4).0.691 0.677 0.694
5).0.567 0.579 0.559
6).0.666 0.677 0.632
7 groups (307): 1) .0.762 0.775 0.757
2).0.658 0.665 0.661
3).0.583 0.586 0.579
4).0.665 0.656 0.668
5).0.757 0.800 0.754
6).0.555 0.548 0.559
8 groups (304): 1) .0.876 0.887 0.854
2).0.958 0.889 0.959
3).0.766 0.749 0.776
4).0.860 0.845 0.866
5).0.879 0.858 0.786
6).0.464 0.455 0.458
9 groups (310): 1) .0.773 0.701 0.690
2).0.879 0.866 0.882
3).0.880 0.800 0.854
4).0.792 0.805 0.811
5).0.669 0.739 0.808
6).0.668 0.678 0.651
10 groups (607): 1) .0.676 0.680 0.733
2).0.968 0.890 0.955
3).0.588 0.586 0.573
4).0.665 0.721 0.776
5).0.887 0.802 0.811
6).0.875 0.885 0.659
11 groups (608): 1) .0.776 0.769 0.717
2).0.658 0.715 0.691
3).0.776 0.768 0.761
4).0.760 0.716 0.776
5).0.687 0.681 0.666
6).0.764 0.786 0.778
12 groups (610): 1) .0.762 0.777 0.775
2).0.589 0.582 0.590
3).0.681 0.684 0.686
4).0.600 0.655 0.661
5).0.878 0.885 0.866
6).0.644 0.654 0.668
13 groups (619): 1) .0.7325 0.697 0.765
2).0.674 0.656 0.682
3).0.880 0.883 0.800
4).0.920 0.895 0.914
5).0.900 0.879 0.886
6).0.688 0.675 0.684
14 groups (623): 1) .0.544 0.587 0.545
2).0.695 0.668 0.659
3).0.557 0.517 0.565
4).0.600 0.645 0.586
5).0.587 0.557 0.499
6).0.564 0.555 0.612
15 groups (631): 1) .0.817 0.855 0.823
2).0.912 0.899 0.887
3).0.576 0.677 0.605
4).0.816 0.824 0.800
5).0.789 0.758 0.715
6).0.590 0.612 0.630
16 groups (634): 1) .0.668 0.612 0.634
2).0.758 0.781 0.796
3).0.666 0.712 0.734
4).0.686 0.684 0.785
5).0.647 0.585 0.703
6).0.844 0.820 0.894
17 groups (639): 1) .1.277 1.101 1.169
2).1.118 1.008 1.082
3).9.880 9.908 9.485
4).9.779 9.811 9.998
5).0.866 0.799 0.818
6).1.266 1.344 1.116
18 groups (640): 1) .1.133 1.112 1.331
2).1.216 1.200 1.228
3).0.771 0.912 0.884
4).0.878 0.833 0.773
5).0.944 0.899 0.912
6).1.441 1.432 1.500
19 groups (645): 1) .0.722 0.754 0.733
2).0.685 0.671 0.657
3).0.814 0.851 0.811
4).0.971 0.918 0.932
5).0.766 0.745 0.789
6).0.866 0.701 0.816
20 groups (647): 1) .1.327 1.310 1.216
2).1.222 1.108 1.130
3).9.999 9.904 9.749
4).1.277 1.311 1.291
5).0.867 0.813 0.828
6).1.126 1.135 1.131
21 groups (654): 1) .0.667 0.736 0.728
2).0.776 0.766 0.752
3).0.977 0.912 0.899
4).0.751 0.713 0.722
5).0.611 0.644 0.601
6).0.876 0.889 0.793
22 groups (656): 1) .0.567 0.568 0.536
2).0.896 0.811 0.895
3).0.658 0.651 0.662
4).0.886 0.821 0.779
5).1.118 0.980 1.198
6).0.769 0.802 0.798
23 groups (657): 1) .1.222 1.198 1.009
2).0.689 0.585 0.599
3).0.768 0.714 0.745
4).0.760 0.672 0.611
5).0.812 0.856 0.825
6).0.649 0.657 0.689
24 groups (658): 1) .0.777 0.768 0.705
2).0.699 0.658 0.643
3).0.581 0.605 0.616
4).0.656 0.678 0.690
5).0.811 0.856 0.844
6).0.764 0.776 0.800
25 groups (659): 1) .1.127 1.111 1.136
2).1.008 1.008 1.032
3).0.780 0.811 0.854
4).1.002 1.238 1.349
5).0.986 0.989 0.811
6).1.126 1.134 1.117
26 groups of mouse (contrast, intramuscular injection 1 μ g recombinant HBsAg/100 μ l PBS)
1).0.441 0.456 0.399
2).0.458 0.473 0.412
3).0.397 0.401 0.423
4).0.500 0.515 0.492
5).0.445 0.413 0.523
6).0.387 0.392 0.328
Background value: 0.311 0.259 0.289
Above-mentioned experimental result shows, the titre of 1-25 group HbsAb generally is higher than 26 groups (contrasts), wherein the highest with 20 groups (647) and 25 groups (659), next is 17 groups (639), 18 groups (640), 22 groups (656) and 23 groups (657) illustrate that CpG ODN has strengthened the immunogenicity of HbsAg, promote the generation of HbsAb.
Embodiment 8: the immune effect of enhanced flow influenza virus vaccine
One. laboratory animal and grouping:
70 7 age in week Kunming mouse, body weight 20 ± 2 gram is divided into 14 groups at random, 5 every group, sub-cage rearing adapts to 2d earlier before the immunity.
Two. immunization route and dosage:
1-13 organizes mouse: intramuscular injection 1 μ g influenza virus vaccine (Changchun Biological Products Institute)/100 μ l PBS adds 10 μ g CpG ODN, divides 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
14 groups: intramuscular injection 1 μ g influenza virus vaccine (Changchun Biological Products Institute)/100 μ lPBS, (no CpG ODN) divides 2 injections, every some injection of promptly two hindlimb muscles 50 μ l.
Three. immune programme for children:
0,21d immunity, for the second time immunity back 7d takes a blood sample through caudal artery, every mouse separates 10ul serum, directly survey antibody titers or put-20 ℃ frozen.
Five, the mensuration of antibody titer (indirect method ELISA)
The reagent equipment:
The bag be cushioned liquid (the 0.05mol/L carbonate buffer solution, pH9.6):
Na 2CO 31.59 gram
NaHCO 32.93 gram
Adding distil water to 1000 milliliter
Lavation buffer solution (PBS-T): contain 0.05% tween 20, pH7.4
K 2HPO 40.2 gram
Na 2HPO 412H 2O 2.9 grams
NaCl 8.0 grams
KCl 0.2 gram
0.5 milliliter of Tween-20
Adding distil water is to 1000ml
Sample diluting liquid:
Bovine serum albumin (BSA) 0.1 gram
Add lavation buffer solution to 100 milliliter
Confining liquid (2%BSA):
Bovine serum albumin (BSA) 2 grams
Add PBS (pH7.4) to 100 milliliters
Substrate buffer solution (phosphoric acid-citrate buffer solution, pH=5.0):
● first liquid (0.1mol/L citric acid)
Citric acid 1.92 grams
Adding distil water to 100 milliliter
● second liquid (0.2mol/L Na 2HPO 4)
Na 2HPO 412H 2O 7.17 grams
Adding distil water to 100 milliliter
Get first liquid 24.3mL, second liquid 25.7mL, adding distil water to 100 milliliter
Tetramethyl biphenyl amine aqueous solution (TMB): 2 mg/ml
10 milligrams of TMB
5 milliliters of ethanol
Substrate solution:
10 milliliters of substrate buffer solutions
0.5 milliliter of TMB (2 mg/ml)
30%H 2O 210 microlitres
Stop buffer:
Dense H 2SO 422ml
Distilled water 178ml
Step
1. bag quilt: being cushioned liquid diluent stream influenza virus vaccine (Chinese Changchun Biological Products Institute provides) to the 1-10 mcg/ml with bag, adding enzyme plate,, spends the night in the 100ul/ hole by 4 ℃.
2. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
3. sealing: add the 2%BSA sealing, the 200ul/ hole.37℃，1.5h。
4. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
5. application of sample: test serum is done 400 times of dilutions, 100ul/ hole with sample diluting liquid.The blank hole adds the sample diluent, the 100ul/ hole, and each sample is established 3 multiple holes.37℃，60min。
6. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
7. add enzyme labelled antibody: rabbit anti-mouse igg-HRP (the biological 0.1ml of Beijing ancient cooking vessel state, 1: 1000) is diluted 500 times with lavation buffer solution, 100ul/ hole, 37 ℃, 60min.
8. washing: wash plate 3 times with lavation buffer solution, 5min/ time.
9. colour developing: add substrate solution, the 100ul/ hole.Room temperature lucifuge reaction 10-30min.
10. stop: 50ul/ hole 2M H 2SO 4
11. microplate reader (BIO-RAD Model 550) is surveyed A 450
Four, result
1-13 organizes mouse (intramuscular injection 1 μ g influenza virus vaccine/100 μ l PBS add 10 μ g CpG ODN)
14 groups of mouse (intramuscular injection 1 μ g influenza virus vaccine/100 μ l PBS)
1 group (205): 1) .0.632 0.598 0.653
2).0.628 0.675 0.634
3).0.528 0.563 0.577
4).0.633 0.701 0.680
5).0.695 0.639 0.629
2 groups (302): 1) .0.723 0.769 0.693
2).0.734 0.786 0.723
3).0.613 0.678 0.645
4).0.636 0.668 0.654
5).0.899 0.864 0.874
3 groups (304): 1) .0.837 0.898 0.829
2).0.676 0.669 0.632
3).0.696 0.637 0.685
4).0.707 0.689 0.670
5).0.887 0.912 0.943
4 groups (310): 1) .0.623 0.659 0.672
2).0.720 0.790 0.728
3).0.767 0.745 0.744
4).0.674 0.614 0.675
5).0.617 0.635 0.677
5 groups (607): 1) .0.872 0.819 0.856
2).0.670 0.635 0.690
3).0.835 0.887 0.876
4).0.67 90.70 50.691
5).0.834 0.825 0.856
6 groups (634): 1) .0.745 0.678 0.677
2).0.634 0.655 0.689
3).0.766 0.746 0.734
4).0.869 0.876 0.821
5).0.734 0.797 0.738
7 groups (639): 1) .0.913 0.945 0.949
2).0.676 0.617 0.690
3).0.782 0.785 0.732
4).1.101 1.206 1.218
5) 0.735 0.795 8 group of .0.768 (640): 1) .0.778 0.821 0.823
2).0.767 0.768 0.753
3).1.008 1.318 1.104
4).1.237 1.134 1.231
5).0.900 0.918 0.922
9 groups (645): 1) .0.887 0.898 0.825
2).0.892 0.875 0.895
3).0.767 0.735 0.723
4).0.785 0.756 0.801
5).0.638 0.776 0.732
10 groups (647): 1) .0.974 0.932 0.998
2).0.912 0.967 1.055
3).0.898 0.998 0.938
4).1.217 1.223 1.021
5).1.105 0.956 0.923
11 groups (656): 1) .0.658 0.644 0.689
2).0.813 0.876 0.874
3).0.768 0.695 0.684
4).0.634 0.612 0.673
5).0.611 0.649 0.673
12 groups (657): 1) .0.769 0.714 0.783
2).0.634 0.705 0.701
3).0.654 0.698 0.708
4).0.763 0.765 0.699
5).0.790 0.745 0.783
13 groups (658): 1) .0.767 0.745 0.798
2).0.761 0.703 0.685
3).0.790 0.804 0.812
4).0.752 0.758 0.782
5).0.688 0.675 0.685
14 groups (659): 1) .0.932 0.978 1.015
2).0.896 0.921 0.943
3).1.007 1.234 0.995
4).0.786 0.723 0.759
5).0.988 0.908 0.954
15 groups (contrast): 1) .0.444 0.428 0.432
2).0.501 0.489 0.473
3).0.376 0.400 0.387
4).0.478 0.465 0.443
5).0.396 0.400 0.412
Background value: 0.301 0.298 0.289.
SEQUENCE?LISTING
＜110〉Changchun Huapu Biotechnology Co., Ltd.
What＜120〉strengthen the protein immune effect of vaccine contains CpG strand deoxy-oligonucleotide
<130>I030020
<160>81
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
<213>Artificial
<400>1
tcgtcgaggg?cgccggtgac 20
<210>2
<211>20
<212>DNA
<213>Artificial
<400>2
tcgtcgccgg?tgggggtgtg 20
<210>3
<211>20
<212>DNA
<213>Artificial
<400>3
tcgtcgtacg?caattgtctt 20
<210>4
<211>20
<212>DNA
<213>Artificial
<400>4
tcgcctcgtc?gccttcgagc 20
<210>5
<211>20
<212>DNA
<213>Artificial
<400>5
tcgcccaccg?gtgggggggg 20
<210>6
<211>21
<212>DNA
<213>Artificial
<400>6
tcgtcgcaga?ccggtctggg?g 21
<210>7
<211>20
<212>DNA
<213>Artificial
<400>7
gggggacgtc?gccggggggg 20
<210>8
<211>20
<212>DNA
<213>Artificial
<400>8
ggatccgtac?gcatgggggg 20
<210>9
<211>20
<212>DNA
<213>Artificial
<400>9
tcgtcgcggc?cggcgccccc 20
<210>10
<211>20
<212>DNA
<213>Artificial
<400>10
tcgtcgcggc?cgcgaggggg 20
<210>11
<211>25
<212>DNA
<213>Artificial
<400>11
tcgtcgttac?cgatgacgtc?gccgt 25
<210>12
<211>26
<212>DNA
<213>Artificial
<400>12
tcgtcgggtg?cgacgtcgca?gggggg 26
<210>13
<211>27
<212>DNA
<213>Artificial
<400>13
tcgtcgggtg?cgacgatcgt?cgggggg 27
<210>14
<211>29
<212>DNA
<213>Artificial
<400>14
tcgtcgtttg?catcgatgca?gtcgtcgtt 29
<210>15
<211>27
<212>DNA
<213>Artificial
<400>15
tcgtcgtttg?catcgatgca?ggggggg 27
<210>16
<211>24
<212>DNA
<213>Artificial
<400>16
accggtatcg?atgccggtgg?gggg 24
<210>17
<211>27
<212>DNA
<213>Artificial
<400>17
ggggtccatg?acgttcctga?agggggg 27
<210>18
<211>26
<212>DNA
<213>Artificial
<400>18
tcgtcgtttt?gacgatcgtc?gggggg 26
<210>19
<211>30
<212>DNA
<213>Artificial
<400>19
ttcgtcgttt?gatcgatgtt?cgttgggggg 30
<210>20
<211>24
<212>DNA
<213>Artificial
<400>20
ttcgtcgttg?tgatcgatgg?gggg 24
<210>21
<211>28
<212>DNA
<213>Artificial
<400>21
tatcgatgtt?ttcgtcgtcg?ttgggggg 28
<210>22
<211>28
<212>DNA
<213>Artificial
<400>22
ttcgttgcat?cgatgcatcg?ttgggggg 28
<210>23
<211>24
<212>DNA
<213>Artificial
<400>23
ttcgcttcgc?ttttcgcttc?gctt 24
<210>24
<211>21
<212>DNA
<213>Artificial
<400>24
tcgaggacaa?gattctcgtg?c 21
<210>25
<211>26
<212>DNA
<213>Artificial
<400>25
tcgaggacaa?gattc?tcgtg?caggcc 26
<210>26
<211>21
<212>DNA
<213>Artificial
<400>26
tcgtgcaggc?caacgaggcc?g 21
<210>27
<211>26
<212>DNA
<213>Artificial
<400>27
accgccaagg?agaagccgca?ggaggg 26
<210>28
<211>16
<212>DNA
<213>Artificial
<400>28
tcgttgccgt?cggccc 16
<210>29
<211>19
<212>DNA
<213>Artificial
<400>29
tacaacggcg?aggaatacc 19
<210>30
<211>30
<212>DNA
<213>Artificial
<400>30
tcggcacgcg?acgtgctggc?cgtcgtttcc 30
<210>31
<211>21
<212>DNA
<213>Artificial
<400>31
gtacaacggc?gaggaatacc?t 21
<210>32
<211>20
<212>DNA
<213>Artificial
<400>32
accgtcgttg?ccgtcggccc 20
<210>33
<211>14
<212>DNA
<213>Artificial
<400>33
tgctggccgt?cgtt 14
<210>34
<211>14
<212>DNA
<213>Artificial
<400>34
gtcggcacgc?gacg 14
<210>35
<211>19
<212>DNA
<213>Artificial
<400>35
gtcggcacgc?gacgggggg 19
<210>36
<211>20
<212>DNA
<213>Artificial
<400>36
gtcggcacgc?gacgcccccc 20
<210>37
<211>22
<212>DNA
<213>Artificial
<400>37
tcgttgccgt?cggccccccc?cc 22
<210>38
<211>19
<212>DNA
<213>Artificial
<400>38
tcgttgccgt?cggcccccc 19
<210>39
<211>18
<212>DNA
<213>Artificial
<400>39
tcgt?tgccgt?cggccccc 18
<210>40
<211>17
<212>DNA
<213>Artificial
<400>40
tcgttgccgt?cggcccc 17
<210>41
<211>20
<212>DNA
<213>Artificial
<400>41
tcgttgccgt?cggccccccc 20
<210>42
<211>13
<212>DNA
<213>Artificial
<400>42
tcgttgccgt?cgg 13
<210>43
<211>14
<212>DNA
<213>Artificial
<400>43
tcgttgccgt?cggg 14
<210>44
<211>15
<212>DNA
<213>Artificial
<400>44
tcgttgccgt?cgggg 15
<210>45
<211>16
<212>DNA
<213>Artificial
<400>45
tcgttgccgt?cggggg 16
<210>46
<211>17
<212>DNA
<213>Artificial
<400>46
tcgttgccgt?cgggggg 17
<210>47
<211>18
<212>DNA
<213>Artificial
<400>47
tcgttgccgt?cggggggg 18
<210>48
<211>19
<212>DNA
<213>Artificial
<400>48
tcgttgccgt?cgggggggg 19
<210>49
<211>20
<212>DNA
<213>Artificial
<400>49
tcgttgccgt?cggggggggg 20
<210>50
<211>19
<212>DNA
<213>Artificial
<400>50
tcgaggacaa?gattctcgt 19
<210>51
<211>14
<212>DNA
<213>Artificial
<400>51
tcccgctgga?cgtt 14
<210>52
<211>27
<212>DNA
<213>Artificial
<400>52
tcggcacgcg?acgtgctggc?cgtcgtt 27
<210>53
<211>21
<212>DNA
<213>Artificial
<400>53
tcgtcgcgcc?gtcacggggg?g 21
<210>54
<211>19
<212>DNA
<213>Artificial
<400>54
tcgtgtgcgt?gccgttggg 19
<210>55
<211>18
<212>DNA
<213>Artificial
<400>55
tcgtcgccgt tgggcggg 18
<210>56
<211>21
<212>DNA
<213>Artificial
<400>56
tcgtcgacgt?cgttgggcgg?g 21
<210>57
<211>26
<212>DNA
<213>Artificial
<400>57
tcgcagttgt?cgtaacgttg?ggcggg 26
<210>58
<211>23
<212>DNA
<213>Artificial
<400>58
ttaccggtta?acgttggccg?gcc 23
<210>59
<211>23
<212>DNA
<213>Artificial
<400>59
accggttaac?gttgtccccg?ggg 23
<210>60
<211>16
<212>DNA
<213>Artificial
<400>60
tcgtcgttgg?tatgtt 16
<210>61
<211>20
<212>DNA
<213>Artificial
<400>61
tcgtcgtcgt?cgttgtcgtt 20
<210>62
<211>24
<212>DNA
<213>Artificial
<400>62
tcgtcgtcgt?cgttgtcgtt?gggg 24
<210>63
<211>15
<212>DNA
<213>Artificial
<400>63
tcgttcgggg?tgccg 15
<210>64
<211>18
<212>DNA
<213>Artificial
<400>64
tcgttcgggg?taacgatt 18
<210>65
<211>17
<212>DNA
<213>Artificial
<400>65
tcgttcgggg?taacgtt 17
<210>66
<211>17
<212>DNA
<213>Artificial
<400>66
tcgttcgggg?taccgat 17
<210>67
<211>21
<212>DNA
<213>Artificial
<400>67
tcgttcgggg?taccgatggg?g 21
<210>68
<211>24
<212>DNA
<213>Artificial
<400>68
tcgttgcgct?cccatgccgg?gggg 24
<210>69
<211>20
<212>DNA
<213>Artificial
<400>69
tcgtcgtttc?gtcgttgggg 20
<210>70
<211>27
<212>DNA
<213>Artificial
<400>70
tcgttgtcgt?ttcgctgccg?gcggggg 27
<210>71
<211>24
<212>DNA
<213>Artificial
<400>71
cgttgacgat?cgtcccatgg?cggg 24
<210>72
<211>16
<212>DNA
<213>Artificial
<400>72
tctgcggcct?tcgtcg 16
<210>73
<211>22
<212>DNA
<213>Artificial
<400>73
tagtaaccgg?tccggcgccc?cc 22
<210>74
<211>19
<212>DNA
<213>Artificial
<400>74
ttgcagcgct?gccggtggg 19
<210>75
<211>23
<212>DNA
<213>Artificial
<400>75
tcgtacggcc?gccgtacggc?ggg 23
<210>76
<211>21
<212>DNA
<213>Artificial
<400>76
cggcccatcg?agggcgacgg?c 21
<210>77
<211>23
<212>DNA
<213>Artificial
<400>77
tcgcgtcgac?tcccctcgag?ggg 23
<210>78
<211>24
<212>DNA
<213>Artificial
<400>78
tcgtcgtcga?ctcgtggtcg?gggg 24
<210>79
<211>20
<212>DNA
<213>Artificial
<400>79
tcgggcgccc?gatcgggggg 20
<210>80
<211>19
<212>DNA
<213>Artificial
<400>80
tcgtcggtct?ttcgaaatt 19
<210>81
<211>18
<212>DNA
<213>Artificial
<400>81
tcgtgacgtc?ctcgagtt 18

Claims (14)

1. biotechnological formulation, it contains strand deoxy-oligonucleotide that contains CpG and protein vaccine that sequence is the synthetic shown in the SEQ ID NO:12, and wherein said protein vaccine is selected from rabies vaccine, hepatitis B vaccine or influenza vaccines.
2. the described biotechnological formulation of claim 1, the phosphodiester bond of wherein said strand deoxy-oligonucleotide is by non-thio-modification, thio-modification or part thio-modification.
3. claim 1 or 2 biotechnological formulation, wherein said strand deoxy-oligonucleotide and described protein vaccine are by the linking agent covalent coupling.
4. sequence is the strand deoxy-oligonucleotide that contains CpG of the synthetic shown in the SEQ ID NO:12 is used for strengthening the biotechnological formulation of protein immune effect of vaccine in preparation application, wherein said protein vaccine is selected from rabies vaccine, hepatitis B vaccine or influenza vaccines.
5. the application of claim 4, the phosphodiester bond of wherein said strand deoxy-oligonucleotide is by non-thio-modification, thio-modification or part thio-modification.
6. claim 4 or 5 application, wherein said biotechnological formulation is before described protein vaccine immunity, use simultaneously behind described protein vaccine immunity or at described protein vaccine immunity, wherein said protein vaccine is selected from rabies vaccine, hepatitis B vaccine or influenza vaccines.
7. sequence is the application of the medicine of the strand deoxy-oligonucleotide that contains CpG of the synthetic shown in the SEQ ID NO:12 differentiation and proliferation that is used for stimulating people's periphery mononuclearcell in preparation.
8. the application of claim 7, the phosphodiester bond of wherein said strand deoxy-oligonucleotide is by non-thio-modification, thio-modification or part thio-modification.
9. sequence is that the strand deoxy-oligonucleotide that contains CpG of the synthetic shown in the SEQ ID NO:12 is used for stimulating people's periphery mononuclearcell to express the application of the medicine of CD19 in preparation.
10. the application of claim 9, the phosphodiester bond of wherein said strand deoxy-oligonucleotide is by non-thio-modification, thio-modification or part thio-modification.
11. being the strand deoxy-oligonucleotide that contains CpG of the synthetic shown in the SEQ ID NO:12, sequence is used for stimulating peripheral blood mononuclear cell to produce the application of the medicine of IL-6 in preparation.
12. the application of claim 11, the phosphodiester bond of wherein said strand deoxy-oligonucleotide is by non-thio-modification, thio-modification or part thio-modification.
13. each application of claim 4-12, wherein said biotechnological formulation is for being suitable for subcutaneous application, and mucomembranous surface is used, and muscle is used, gastrointestinal applications, the form that use in the abdominal cavity.
14. each application of claim 4-12, wherein said biotechnological formulation and adjuvant combined utilization, described adjuvant is selected from aluminium adjuvant or freund's adjuvant.
CNB031198414A 2003-03-05 2003-03-05 Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine Expired - Lifetime CN100439386C (en)

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EP1829887B1 (en) 2004-11-29 2011-08-24 Changchun Huapu Biotechnology Co., Ltd. Cpg single-strand deoxynucleotides for use as adjuvant
CN1810970B (en) * 2005-01-27 2011-05-18 长春华普生物技术有限公司 CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application
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CN105936906B (en) * 2013-11-08 2019-06-21 上海交通大学 Oligodeoxynucleotide molecule for the sequence units containing CpG being modified and application thereof
CN106893724B (en) * 2015-12-17 2023-05-02 苏州派动生物技术有限公司 Oligonucleotide with antigen synergism and tumor treatment effect
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