CN103789315A - PEGylated CpG oligonucleotide and application thereof - Google Patents

Abstract

The invention relates to PEGylated CpG oligonucleotide and application of the PEGylated CpG oligonucleotide in the preparation of a medicament for improving the IL-12 expression quantity in serum, a medicament for inducing the immunogenicity to an endogenous antigen such as a tumor antigen and a medicament for stimulating the immunocompetence of B cells, plasmacytoid dendritic cells, myeloid dendritic cells, macrophages and peripheral mononuclear cells in the field of vaccine and tumor immunotherapy administration systems. The PEGylated CpG oligonucleotide has the effects of homeostasis improvement and immunoregulation, and response to persistent antigens in vivo can be improved.

Description

CpG oligonucleotide and application thereof that PEG modifies

Technical field

The invention belongs to vaccine and immunotherapy of tumors drug delivery system field, be specifically related to CpG oligonucleotide and application thereof that a kind of PEG modifies.

Background technology

CpG oligodeoxynucleotide (oligodeoxynucleotides, ODN) be some synthetic to there is CpG sequence (cytosine(Cyt)-phosphoric acid-bird pyrimidine) motif of immune activation function as the oligodeoxynucleotide of core, its constitutional features is 5 '-PuPuCPGPyPy-3, both 5 ' end had two purine or a purine to connect a thymus pyrimidine, and 3 ' end has the oligonucleotide sequence of two pyrimidines.

In the nineties in 19th century, U.S. tumour doctor Coley is by bacterium crude extract during for the treatment of 900 long-term tumour patients, finds that there is patient's tumour of 40% and disappears voluntarily.At that time, it is believed that it is that lipopolysaccharides wherein plays a role, and do not cause enough attention.Afterwards, while carrying out mouse in vivo test with bacille Calmette-Guerin vaccine, to find, the bacille Calmette-Guerin vaccine that DNA enzyme was processed does not have antitumor action, and this explanation DNA of bacteria is only the medicine that antitumor action occurs.Afterwards, studies confirm that, DNA of bacteria has directly antitumor and immunostimulating effect.Nineteen ninety-five, Krieg etc. find that, in DNA of bacteria, what stronger immunization occurred is CpG motif, and this class sequence is become to immunostimulatory sequence.Different from bacterium, in the DNA molecular of Mammals and other animals, the frequency of occurrences of CpG motif is very low, and in the CpG motif of 60%-90%, the 5th carbon atom of cytosine(Cyt) methylates simultaneously, because this variation, mammiferous DNA molecular does not have immunostimulation.CpG motif is the basic structure that CpG DNA produces immunostimulation function, among one section of synthetic CpG ODN, can there be a motif or multiple CpG motif, purine and the pyrimidine of CpG motif both sides and form the several bases between different motifs, all can affect the immunostimulation function of CpG motif.About studies show that in early days of the structure of CpG ODN, the immunization of CpG motif has species specificity, for example, be 5'-GACGTT-3' to the most effective CpG motif of mouse, and be 5'-GTCGTT-3' to the most effective CpG motif of people.According to CpG ODN structure and the difference to human peripheral blood mononuclear cell (peripheral blood mononuclear cell, PBMC) hormesis in vitro, it is generally acknowledged that a CpG ODN is divided into 3 classes: A type, Type B, and C type.A type CpG ODN can stimulate a large amount of interferon alphas of generation (IFN-α), and can activate in a large number natural killer cell (natural killer, NK); Type B CpG ODN can stimulate B cell proliferation to secrete a large amount of immunoglobulin (Ig)s (Immunoglobulin, IgG), interleukin-6 (Interleukin-6, IL-6) and IL-10 (Interleukin-10, IL-10) simultaneously; C type CpG ODN has the effect feature of A type and Type B CpG ODN concurrently.

The oligodeoxynucleotide (CpG ODN) that contains non-methylated CpG motif can be identified by intracellular Toll sample receptor 9 (TLR9), activate Th1 immunne response to strengthen intrinsic and acquired immunity, on the one hand, CpG ODN has activated TLR9 signal induction, and the interior signal pathway of nuclear factor and other cell activates, start quick innate immune reaction, comprise the secretion of multiple proinflammatory cytokine and antiviral cell factor, as IL-6, tumor necrosis factor-alpha (TNF-α) and I type Interferon, rabbit (IFN); And the secretion of immunomodulating cytokines, as IL-10, with the intensity of the reaction that controls inflammation, and the approach that NK cell and other innate immune cells rely on by IFN Dependent or non-IFN is by indirect activation.On the other hand, the B cell of CpG ODN activation improves greatly to the susceptibility of antigenic stimulation, accelerates to be divided into the plasmocyte of secretion antigen specific antibody, thereby has promoted Acquired immune response; The pDC that CpG ODN activates is to the T cellular regions migration of lymphoglandula and other secondary lymphoid tissue, expressing costimulatory molecules level raises, its ability that activates T cells and memory t cell is strengthened, and the ability that pDC offers Soluble protein antigen to cd8 t cell intersection strengthens, the CD4 of Th1 approach and the immunne response of cd8 t cell are strongly promoted to rely on.It has very wide application prospect as immunological adjuvant in antiviral and antineoplaston field to CpG at present.

But since upper world CpG nineties ODN finds to have effect that activate immunity replys so far, the but CpG marketing drugs of neither one maturation still.The CpG7909 of Pfizer stops at the conceptual phase of clinical three phases, be mainly due to CpG7909 combine with chemical drug use treatment nonsmall-cell lung cancer do not increase curative effect, the case that these are failed, has embodied the defect on existing CpG medicines structure.Our data show that common CpG ODN and the conventional stability or very poor in serum of the modification structure on phosphodiester bond skeleton connect PEG and contributes to greatly to improve its serum stability.But people it is generally acknowledged, and having lot of documents support, the PEG meeting of modification hinders containing the molecule of PEG and the interaction of immunocyte, particularly according to bibliographical information, the acceptor TLR9 of CpG is mainly distributed in endocytosis body inner side, so the CpG ODN that PEG modifies will no longer have immunocompetence.But we find by research, the CpG ODN that PEG modifies can not only act on B cell and DC emiocytosis cytokine, can also improve in body the concentration of IL-12 in serum, is also better than even in vivo the CpG of unmodified in experiment.

In addition, PEG modifies (PEGylation) although be successfully applied to the modification of multiple protein molecule and nano medicament carrying system, can greatly improve serum stability and the transformation period of albumen, but be almost seldom used for modified oligonucleotide (ODN) molecule, comprise siRNA, microRNA, Cpg ODN etc., the action target spot that major cause is ODN is mostly in cell, and the ODN molecular weight of modifying through PEG is larger, and the efficiency that enters cell should be lower.The method of more favoring in the industry, is to utilize some nano medicament carrying systems, protects the stability of ODN, improves cellular uptake.But we find, for specific CpG ODN series sequence modification PEG, can't block it and enter cell generation immunostimulation, even effect is also improved in vivo, thereby has disclosed new mechanism and the new direction of CpG ODN as anti-tumor medicine.

Summary of the invention

The present invention realizes by following technical scheme, the present invention relates to CpG oligonucleotide and application thereof that a kind of PEG modifies.The present invention relates to the CpG that PEG modifies; There is body internal stability and the immunoloregulation function of increasing, can improve in vivo the responsing reaction for the antigen of sustainable existence.

The present invention realizes by following technical scheme,

First aspect, the present invention relates to the CpG oligonucleotide that a kind of PEG modifies.

Preferably, mw >=10K of described PEG.

Preferably, the skeleton of described oligonucleotide is full thio-modification, part thio-modification or without thio-modification.

Preferably, the site that described PEG modifies is 5 '-end or 3 '-end.

Preferably, described structure is shown in formula 1 or formula 2, the CpG unit that described OLIGO oligonucleotide wherein comprises 1-5 unit;

Preferably, the sequence of described CpG oligonucleotide is:

The CpG-7 of sequence as shown in SEQ ID NO.1,

The CpG-8 of sequence as shown in SEQ ID NO.2,

The CpG-13 of sequence as shown in SEQ ID NO.3,

The CpG1826 of sequence as shown in SEQ ID NO.4,

The CpG7909 of sequence as shown in SEQ ID NO.5,

The CpG-H7 of sequence as shown in SEQ ID NO.6,

The CpG-H8 of sequence as shown in SEQ ID NO.7,

The CpG-H13 of sequence as shown in SEQ ID NO.8,

The CpG-H1826 of sequence as shown in SEQ ID NO.9.

Preferably, the CpG oligonucleotide that described PEG modifies can be synthesized into by the method comprising the steps;

Route a, joins the CpG of sulfydryl modification in Tris-HCL, blows N ₂5min, then add three (2-carbonyl ethyl) microcosmic salt hydrochlorate, mix, then add mPEG-Mal, N in whole process ₂protection, vortex mixes, and 25 ℃, 500-1300rpm reacts 5h; By the mixture dichloromethane extraction of the product obtaining, collect dichloromethane layer solution, repeatedly extract three times, volatilize with nitrogen, add ultrapure water to dissolve; 4 ℃ of dialysed overnight of dialysis tubing of finally obtained synthetic product being made with the dialysis membrane of 3500Da, dialysis finishes sample freeze-drying, to obtain final product;

Route b, the mPEG that alkynyl-modified CpG and nitrine are modified, in the reaction solvent of being prepared for 1:1 by volume by water and methyl alcohol, does at cupric sulfate pentahydrate and sodium ascorbate under the condition of catalyzer and vibrates and mix, and reacts 18h and get final product with 37 ℃;

Second aspect, the CpG oligonucleotide that the present invention relates to a kind of modification of PEG as the aforementioned improves the application in the IL-12 expression amount medicine in serum in preparation.

The third aspect, the CpG oligonucleotide that the invention still further relates to a kind of modification of PEG is as the aforementioned induced for endogenous antigen as the application in the immunogenicity medicine of tumour antigen in preparation.

Fourth aspect, the invention still further relates to a kind of CpG oligonucleotide of the modification of PEG as the aforementioned in the application of preparing in the immunocompetence drug that stimulates B cell, plasmacytoid dendritic cells, medullary system dendritic cell, scavenger cell, peripheral mononuclear cells.

The present invention has following beneficial effect:

The present invention relates to the CpG that PEG modifies; There is the body internal stability of increasing and immunoloregulation function;

The concrete PEG that adopts 10K-20K chain length modifies the oligonucleotide molecules that contains CpG sequence, although PEG modifies, its hormesis to mDC and splenocyte is decreased, but can improve in vivo the responsing reaction for the antigen of sustainable existence, particularly for the endogenic tumour antigen of tumor-bearing mice;

PEG modifies on the one hand can improve IL-12, improves immunne response, can also reduce the action effect of CpG for regulatory T cells (Treg), significant in immunotherapy of tumors.

PEG is modified at the cellular uptake to CpG ODN and targeted integration to a certain extent certain influence, but still has good immunostimulation and dose-effect relationship; PEG modifies the effect that can play action time in extension body, the immunne response better effects if that the structure after modification excites in vivo, and antitumous effect is stronger;

The circulative raising of length that CpG is administered systemically, it is reacted synthetic with the CpG ODN with immunoloregulation function by PEG; Thereby this combination can effectively improve the body internal stability of CpG ODN and improve its immunoloregulation function in vivo, in order to strengthen immunne response;

The immunization of CpG motif has species specificity, for example, be 5'-gacgtt-3' to the most effective CpG motif of mouse, and be 5'-gtcgtt-3' to the most effective CpG motif of people; According to known theory to the sequence of mouse sensitivity as changed the responsive sequence of adult, as CpG-7 (tgacgttccgtt) is converted to for the effective sequence C pG-H7 of people (atgtcgttgcgtt), be used for anaphylactic disease, communicable disease, immunodeficiency diseases and cancer etc. as medicine more effectively.

Accompanying drawing explanation

By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:

Fig. 1. the impact of differential responses time on synthetic ratio;

Fig. 2. different CpG and the feed ratio of PEG affect agarose electrophoresis figure to reaction efficiency;

Fig. 3 .CpG-PEG synthetic route chart;

The agarose gel electrophoresis image of Fig. 4 .CpG-PEG;

High performance liquid phase (HPLC) color atlas of Fig. 5 .CpG and CpG-PEG;

The infrared spectrogram of Fig. 6 .DCC reaction;

Fig. 7. cut glue and reclaim the SDS-PAGE electrophoretic image of rear three kinds of different molecular weight PEG-CpG;

Fig. 8. the external immunostimulating effect of different chain length CpG;

Fig. 9. the external immune effect difference of CpG-PEG of different chain length;

Figure 10. the 14th day mice serum IgG concentration after initial immunity;

Figure 11. the 14th day mice serum IgG1 concentration after initial immunity;

Figure 12. the 14th day mice serum IgG2a concentration after initial immunity;

Figure 13. the 14th day mice serum IgG2a/IgG1 concentration ratio after initial immunity;

Figure 14. the 28th day mice serum IgG concentration after initial immunity;

Figure 15. the 28th day mice serum IgG1 concentration after initial immunity;

Figure 16. the 28th day mice serum IgG2a concentration after initial immunity;

Figure 17. the 28th day mice serum IgG2a/IgG1 concentration ratio after initial immunity;

Figure 18. the dynamic change of mice serum IgG2a concentration;

Figure 19. the dynamic change of mice serum IFN-γ concentration;

Figure 20. different concns CpG and CpG-PEG affect 4T1-pGL3 cytoactive;

Figure 21 .CpG-PEG stimulates the concentration of BMDC emiocytosis IL-12p70;

Figure 22. different dosing group mouse tumor growth curve;

Figure 23. the dynamic change of tumor-bearing mice serum IL-12p70.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The test method of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Pehanorm Brooker equimolecular clone: the condition described in the laboratory manual third edition (Science Press, 2002), or the condition of advising according to each manufacturers.

embodiment 1, the impact of differential responses time on structure 1 synthetic ratio

(1) after ultrapure water boils, pass into N ₂after cooling, with deoxygenation, be mixed with pH=7.4 with this ultrapure water, the Tris-HCl of 10mM is as reaction solvent.The CpG-SH that gets 20nmol is dissolved in the Tris-HCl of 10mM of 100ul, after dissolving, blows N ₂5min clock.Take the TCEP of 3.5mg, add the 10mM Tris-HCL (pH=7.4) of 4ml, obtain the TCEP into 3nmol/ μ l, blow 5min with nitrogen, draw 10ul in the EP pipe of CPG-SH, mix.Take the mPEG of 1.0mg _20Kother molecular weight of-mal(increase or reduce with equal proportion) be dissolved in CpG-SH solution nitrogen protection in whole process.On constant temperature oscillation instrument 25 ℃, 800rpm reacts respectively 5h, 10h, 15h, 25h.

(2) sample (1) being obtained adopts the relatively difference of synthetic ratio of agarose gel electrophoresis, the results are shown in Figure 1, in Fig. 1, band order is from left to right: DNA marker, CpG, reaction times is the reaction product of 5h, reaction times is the reaction product of 10h, the reaction product that the reaction times is 15h, the reaction product that the reaction times is 20h.。Experimental result shows: the reaction times is little on synthetic ratio impact, considers the property of experiment, and the selective reaction time is 5h in time.

the impact of the feed ratio of embodiment 2, CpG-PEG and CpG on structure 1 reaction yield

(1) after ultrapure water boils, pass into N ₂after cooling, with deoxygenation, be mixed with pH=7.4 with this ultrapure water, the Tris-HCl of 10mM is as reaction solvent.The CpG-SH that gets 15nmol is dissolved in the 10mMTris-HCl of 20ul, after dissolving, and nitrogen blowing 5min clock.Take the TCEP of 3.5mg, add the 10mMTris-HCl (pH=7.4) of 4ml, obtain the TCEP into 3nmol/ μ l, blow 5min with nitrogen, draw 10ul in the ep pipe of CPG-SH, mix.And be packed as 3 pipes, take respectively 0.2mg, 0.4mg, the mPEG of 0.6mg _20K-mal (other molecular weight increase or reduce with equal proportion) is dissolved in CpG-SH solution, vortex vibration.Nitrogen protection in whole process.On constant temperature oscillation instrument 25 ℃, 800rpm reacts respectively 5h.

(2) sample (1) being obtained adopts the relatively difference of synthetic ratio of agarose gel electrophoresis, the results are shown in Figure 2, (band order is from left to right: the DNA marker of 20bp, CpG, reactant molar ratio example is the product of 1:2, the product that reaction molar ratio is 1:6, the product of reactant molar ratio example 1:12) conclusion: reactant molar ratio is that 1:6 and 1:12 are higher than the combined coefficient of 1:2, combined coefficient can reach 80%, but the difference of 1:6 and 1:12 is little, so consider to use the reaction ratio of 1:6 in subsequent experimental.

the method for synthesizing and purifying of CpG-PEG shown in embodiment 3, structure 1

1.CpG-PEG _20Ksynthetic method (Fig. 3).

(1) after ultrapure water boils, pass into N ₂after cooling, with deoxygenation, be mixed with pH=7.4 with this ultrapure water, the Tris-HCL of 10mM is as reaction solvent.The CpG-SH that gets 20nmol is dissolved in the Tris-HCL of 10mM of 100ul, after dissolving, and nitrogen blowing 5min clock.Take the TCEP of 3.5mg, add the 10mMTris-HCL (pH=7.4) of 4ml, obtain the TCEP into 3nmol/ μ l, blow 5min with nitrogen, draw 10ul in the EP pipe of CPG-SH, mix.The mPEG20K-mal (other molecular weight increase or reduce with equal proportion) that takes 1.6mg adds in above-mentioned solution, and vortex vibration, dissolves it completely.N in whole process ₂protection.On constant temperature oscillation instrument 25 ℃, 800rpm reacts respectively 5h.Can zoom in or out in proportion dosage synthetic.

(2) mixture obtaining after the reaction (1) being obtained adopts the ratio extraction solution of volume ratio for (mixture: methylene dichloride)=1:10, obtains the solution of layering, and dichloromethane layer is PEG and CpG-PEG _20kmixture, water layer is the CpG that has neither part nor lot in reaction, 4 ℃, 10000rpm is centrifugal, to remove emulsion, take out dichloromethane layer, re-extract 3 times, volatilizes the dichloromethane layer obtaining with nitrogen, add ultrapure water dissolve.

(3) 4 ℃ of dialysed overnight of dialysis tubing that the synthetic product (2) being obtained is made with the dialysis membrane of 3500Da, unnecessary impurity is removed in dialysis.Obtain the mix products of CpG-PEG and PEG.Sample freeze-drying after dialysis is beneficial to preserve.

The characterizing method of 2.CpG-PEG, has comprised agarose gel electrophoresis and high performance liquid chromatograph.

(1) agarose gel electrophoresis related experiment parameter is as follows:

Agarose gel electrophoresis characterizes: the agarose of 0.75-3%, damping fluid: TBE, DNA marker:20bp, applied sample amount: 100ng-600ng, fluorescence dye: Gelred.With this understanding, can be by CpG and CpG-PEG _20ksignificantly make a distinction.Synthetic ratio can reach 80% left and right.(Fig. 4, band order is from left to right: the DNA marker of 20bp, CpG, unpurified synthetic product CpG-PEG, the synthetic product CpG-PEG of purifying)

(2) the related experiment parameter that high performance liquid chromatography (HPLC) characterizes is as follows:

The high performance liquid chromatography testing conditions of CpG-PEG is: moving phase adopts acetonitrile and 100mMTEAA (pH=6), and gradient is: acetonitrile: 5-65%(0-15min), 65%-5%(15-20min); Chromatographic column: ZORBAX300SB-C18 chromatographic column (200mm × 4.6mm, 5 μ m, Agilent, Technologies); Agilent 1200 high performance liquid chromatographs; Detect wavelength: 260nm; Flow velocity 1mL/min; Sample size: 10 μ L.The results are shown in (Fig. 5), with this understanding, good separating effect, peak shape symmetry, appearance time is short, and retention time is stable.

cpG-PEG synthetic method shown in embodiment 4, structure 2

The different molecular weight PEG that the synthetic nitrine of 1.DCC reaction is modified

Experiment reagent: DCC (1,3-dicyclohexylcarbodiimide), DMAP(4-Dimethylamino pyridine), mPEG.

Reaction mechanism: this reaction utilizes DCC to make the hydroxylic moiety of PEG and the carboxy moiety dehydrating condensation of nitrine acetic acid, makes PEG connect smoothly upper azido-, for ensuing click reaction is prepared.DMAP adds DMAP in reaction, also can make the efficiency of reaction further improve as a kind of catalyzer of high nucleophilicity through exploration discovery.

(1) reaction reagent bottle used etc. is put into vacuum drying oven and dried stand-byly, guarantee the anhydrous condition in whole reaction.

(2) get dried molecular sieve and put into the reagent bottle that methylene dichloride is housed, guarantee the anhydrous condition of methylene dichloride.

(3) prepare nitrogen protection capsule.

(4) mPEG and nitrine acetic acid amount are as requested weighed up fast, then add rapidly in the methylene dichloride of 25ml, affect reaction to reduce reagent to absorb airborne moisture.

(5) add DCC, reaction system is put on magnetic stirring apparatus and is stirred, in not adding DMAP, just start muddiness.In the time adding DMAP, solution becomes faint yellow at once, and occurs muddy.

(6) now nitrogen gas cell is pricked on the round-bottomed flask of reaction, at normal-temperature reaction 48h.

(7) react after 48h stopped reaction.

(8) adopt Rotary Evaporators that methylene dichloride is removed.

(9) product is dissolved in water, filters.

(10) product of reaction is put in dialysis tubing, by dialysis clamp clamping, dialysed overnight.

(11) sample after dialysis is put in Freeze Drying Equipment and is dried.

2. infrared spectra detects

Operation steps

(1) detection of instrument:

Test wave-number range: 4000～400cm-1 wave number precision :≤0.1cm-1 resolving power: 0.1～16cm ^-1.Instrument service voltage: 220V ± 10%, frequency 50Hz ± 10%, the environment at instrument place: room temp is that 20 degrees Celsius of relative humidity are 30% left and right.

(2) preparation of sample

Get 1 to the unreacted PEG sample of 2mg, add appropriate Potassium Bromide and be put in agate mortar and grind to form fine powder, pack in mould, on tabletting machine, be pressed into the sheet of transparence, for check.

(3) step of repetition (2) is prepared other trial-product and reference substance

(4) detect sample and check out peak situation (Fig. 6).

Note: (a) be depicted as the not PEG through reaction _5K

(b) PEG for having reacted _5K

Contrast two pictures and can find that 2107 places in figure b have the peak position of azido-to occur proved response success.

(c) be depicted as the not PEG through reaction _10K

(d) PEG for having reacted _10K

Contrast two pictures and can find that 2107 places in figure d have the peak position of azido-to occur proved response success.

(e) be depicted as the not PEG through reaction _20K

(f) PEG for having reacted _20K

Contrast two pictures and can find that 2107 places in Fig. 2 have the peak position of azido-to occur proved response success.

Known by above infrared result, DCC reaction has successfully been linked nitrine group on the PEG of different molecular weight, simultaneously, because next nitrine PEG belongs to the excessive side of reaction, so gained nitrine is modified to the not too large requirement of purity of PEG, therefore do not need purified step, but must be except the by product of dereaction.Take dialysis and cryodesiccated method to carry out processing sample here.

The CpGODN that the synthetic PEG of 3.Click reaction modifies

(1) the first PEG that accurately takes 8mg _20K(other molecular weight increase or reduce with equal proportion), the cupric sulfate pentahydrate of 6mg, the sodium ascorbate of 4.75mg, be put in respectively in different ep pipes, and carry out mark.

(2) cupric sulfate pentahydrate of 6mg is dissolved in 1ml water and gets 25ul, be dissolved in and in 1ml water, get 250ul and mutually mix with 4.75mg sodium ascorbate.

(3) load weighted PEG is dissolved in to the 100ul water that proportioning is good: in 100ul methanol mixed solvent.Get the catalyzer 27.5ul mixing and join in PEG, be put in vibrator and mix.

(4) CpG of 20ul is dissolved among PEG and concussion mixes, in 37 ℃ of reaction 18h.

4. polyacrylamide gel electrophoresis confirmatory reaction product

(1) urea of glue raw material and consumption: 4.2g, 1mlTBE damping fluid, 4.44ml acrylamide, 40ulAP, 4ulTBMB.When noting glue, there is not urea to separate out.

(2) glue preparing is added in off-the-shelf sheet glass fast, treat 1h left and right, wait gelling well afterwards to add TEA damping fluid, added after damping fluid, guarantee that damping fluid can not spill, loading afterwards.Can prepare sample Deng glue solidificating period.After having gone up sample, open electrophoresis apparatus, start to run glue, after about two or three hours, sample, to time colloid edge, stops electrophoresis.Glue is taken off, put into watch-glass, add staining agent supergreen to start dyeing, when approximately dying about half hour, glue is taken out, develop.

5. cut glue and reclaim, operation steps:

(1) in the parting line zone of moistening gel, cut out the gel fragment that contains object band, attention will make the gel fragment that cuts thin as much as possible, avoids sneaking into Unspccific bands.While cutting glue, should use disposable surgical blade, prevent DNA crossed contamination.

(2) band cutting is placed in to the ep pipe that 20mlTE liquid is housed, gel is fully pulverized to rear vibration wash-out, be placed in 56 ℃ of baking boxs and spend the night with sealed membrane sealing

(3) take out and be placed on 4 ℃ of refrigerator and cooled but, after the centrifugal 3min of 12000rpm, get supernatant liquor.

6. sample preparation

Because the CpGODN modifying through PEG contains a large amount of metal ions in its reaction solution, can not be directly used in injection mouse, need to enter dialysis, freeze-drying, then the process of dissolving, just can carry out the experimentation on animals of safety non-toxic.Its molecular weight of CpGODN of modifying through PEG is greater than 10, 000Da, the molecular weight of CpGODN monomer also has 6, 500Da, and contained organic reagent, the molecular weight of heavy metal ion is all less than 1, 000Da, therefore we to select molecular weight cut-off (staying the minimum molecular weight of the biomacromolecule in dialysis tubing) be 3, the dialysis membrane of 500Da, clip 2cm length, one end clamps with dialysis clamp, make a dialysis tubing, then the CpGODN reaction solution of PEG being modified adds in dialysis tubing, then also clamp with dialysis clamp at dialysis tubing opening end, put into the large beaker that fills with ultrapure water, 4 ℃ are stirred dialysis 6h.

difference between embodiment 5, CpG-PEG that relatively different modifying method is right

1. external collection and the cultivation of splenocyte: get one of 8 weeks Balb/c mouse, de-neck method is put to death, 75% alcohol-pickled 5min.Dissect mouse, open abdominal cavity, take out spleen.Spleen is put in to cell sieve upper, adds PBS, grind.Suction pipe is collected the liquid after grinding, and adds centrifuge tube, 4 ℃ of centrifugal 5min of 1400rpm.After centrifugal, abandoning supernatant, adds 5ml erythrocyte cracked liquid, resuspended, and 4 ℃ 1400 leaves the heart 5 minutes, abandoning supernatant, and repeating this step to pipe floor cells is white.Use 1640 substratum, re-suspended cell, draws 10 μ l and splashes into cell counting count board, cell counting under microscope.Cell is joined to 96 orifice plates.96 orifice plates are positioned over to 37 ℃, 5%CO ₂in incubator, cultivate respectively 2h.

2. sample solution preparation: the solution of using the CpG-PEG of the different modifying method that PBS compound concentration is identical.

3. cell administration: the sample solution that takes out different concns joins in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 degree, CO ₂concentration 5% is cultivated after 24h, and 4 ℃, the centrifugal 5mim of 1400rpm, collects supernatant liquor and measure for ELISA.

4. result: experiment in vitro shows that different modifying method does not have significant difference to CpG stimulated in vitro effect.

the external immune effect difference of CpG ODN sequence of embodiment 6, comparison different chain length

1. external collection and the cultivation of splenocyte: get one of 8 weeks Balb/c mouse, de-neck method is put to death, 75% alcohol-pickled 5min.Dissect mouse, open abdominal cavity, take out spleen.Spleen is put in to cell sieve upper, adds PBS, grind.Suction pipe is collected the liquid after grinding, and adds centrifuge tube, 4 ℃ of centrifugal 5min of 1400rpm.After centrifugal, abandoning supernatant, adds 5ml erythrocyte cracked liquid, resuspended, 4 ℃ of centrifugal 5min of 1400rpm, and abandoning supernatant, repeating this step to pipe floor cells is white.Use 1640 substratum, re-suspended cell, draws 10 μ l and splashes into cell counting count board, cell counting under microscope.Join in 96 orifice plates.96 orifice plates are positioned over to 37 ℃, 5%CO ₂in incubator, cultivate respectively 2h.

2. sample solution preparation: with the solution of the identical CpG as shown in the table of PBS compound concentration.

Group	Sequence (5 '-3 ')	Sequence numbering (SEQ ID NO.)
			CpG1826	tccatgacgttcctgacgtt		9
CpG-1	atggacgttcg	10
			CpG-2	atagacgttcg	11
CpG-3	atgaacgttcg	12
			CpG-4	tcagacgttcg	13
CpG-5	tcaaacgttcg	14
			CpG-6	gacgttcccgtt	15
CpG-7	tgacgttccgtt	1
			CpG-8	atgacgttgcgtt	2
CpG-9	tcgcggtcgttcgcg	16
			CpG-10	ttgtcgttcgcg	17
CpG-11	gtcgttcgcg	18
			CpG-12	tccatgacgttcc	19
CpG-13	tccatgacgttcctgacgttcctgacgtt	3
			CpG7909(CpG2006)	tcgtcgttttgtcgttttgtcgtt	5

3. cell administration: take out sample solution and join in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 degree, CO2 concentration 5% was cultivated after 24 hours, and 4 ℃, the centrifugal 5min of 1400rpm, collects supernatant liquor and measures for ELISA.

4. result: can find out by upper result, the CpG of different chain length stimulates splenocyte secretion IL-6 there are differences, compared with blank group, CpG1826, CpG-7, CpG-8, CpG-13, CpG7909 (CpG2006) stimulates the expression of mouse boosting cell secretion IL-6 to have showing property to improve (Fig. 8).

the difference of the CpG ODN epi sequence effect of embodiment 7, more different backbone modifications

1. external collection and the cultivation of splenocyte: get one of 8 weeks Balb/c mouse, de-neck method is put to death, 75% alcohol-pickled 5min.Dissect mouse, open abdominal cavity, take out spleen.Spleen is put in to cell sieve upper, adds PBS, grind.Suction pipe is collected the liquid after grinding, and adds centrifuge tube, 4 ℃ of centrifugal 5min of 1400rpm.After centrifugal, abandoning supernatant, adds 5ml erythrocyte cracked liquid, resuspended, and 4 ℃ 1400 leaves the heart 5 minutes, abandoning supernatant, and repeating this step to pipe floor cells is white.Use 10ml1640 substratum, re-suspended cell, draws 10 μ l and splashes into cell counting count board, cell counting under microscope.Add in 96 orifice plates, 96 orifice plates are positioned over to 37 ℃, in 5%CO2 incubator, cultivate respectively 2h.

2. sample solution preparation: use the solution of the CpG of the different backbone modifications that PBS compound concentration is identical, comprise full thio-modification, part thio-modification, without the CpG. of thio-modification

3. cell administration: take out the sample solution 5 μ l of different concns, join in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 ℃, CO ₂concentration 5% is cultivated after 24h, and 4 ℃, the centrifugal 5mim of 1400rpm, collects supernatant liquor and measure for ELISA.

4. result: can find out by upper result, the CpG of different backbone modifications stimulates splenocyte secretion IL-6 there are differences, and the effect of stimulation of full thio-modification group is best.But compared with blank group, the CpG of different backbone modifications stimulates the expression of splenocyte secretion IL-6 to have showing property to improve.

the difference of the CpG-PEG oligonucleotide sequence immune effect of embodiment 8, more different backbone modifications

1. external collection and the cultivation of splenocyte: get one of 8 weeks Balb/c mouse, de-neck method is put to death, 75% alcohol-pickled 5 minutes.Dissect mouse, open abdominal cavity, take out spleen.Spleen is put in to cell sieve upper, adds PBS, grind.Suction pipe is collected the liquid after grinding, and adds centrifuge tube, 4 ℃ of centrifugal 5min of 1400rpm.After centrifugal, abandoning supernatant, adds 5ml erythrocyte cracked liquid, resuspended, 4 ℃ of centrifugal 5min of 1400rpm, and abandoning supernatant, repeating this step to pipe floor cells is white.Use 10ml1640 substratum, re-suspended cell, draws 10 μ l and splashes into cell counting count board, cell counting under microscope.Cell is joined in 96 orifice plates, be positioned over 37 ℃, 5%CO ₂in incubator, cultivate respectively 2h.

2. sample solution preparation: use the solution of the CpG-PEG of the different backbone modifications that PBS compound concentration is identical, comprise the CpG-PEG of full thio-modification, part thio-modification CpG-PEG, without the CpG-PEG. of thio-modification

3. cell administration: take out sample solution and join in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 ℃, CO ₂concentration 5% is cultivated after 24h, and 4 ℃, the centrifugal 5min of 1400rpm, collects supernatant liquor and measure for ELISA.

4. result: can find out by upper result, the CpG-PEG of different backbone modifications stimulates splenocyte secretion IL-6 there are differences, and the effect of stimulation of the CpG-PEG of full thio-modification is best.But compared with blank group, the CpG-PEG of different backbone modifications stimulates the expression of splenocyte secretion IL-6 to have showing property to improve.

embodiment 9, the external immunocompetent difference of CpG that relatively PEG of different chain length modifies

1. external collection and the cultivation of splenocyte: get one of 8 weeks Balb/c mouse, de-neck method is put to death, 75% alcohol-pickled 5 minutes.Dissect mouse, open abdominal cavity, take out spleen.Spleen is put in to cell sieve upper, adds PBS, grind.Suction pipe is collected the liquid after grinding, and adds centrifuge tube, 4 ℃ of centrifugal 5min of 1400rpm.After centrifugal, abandoning supernatant, adds 5ml erythrocyte cracked liquid, resuspended, centrifugal 5 minutes of 4 ℃ of 1400rpm, and abandoning supernatant, repeating this step to pipe floor cells is white.Use 10ml1640 substratum, re-suspended cell, draws 10 μ l and splashes into cell counting count board, cell counting under microscope.Cell is joined in 96 orifice plates, be positioned over 37 ℃, 5%CO ₂in incubator, cultivate respectively 2h.

2. sample solution preparation: with the identical CpG of PBS compound concentration, CpG-PEG _5K, CpG-PEG _10K, CpG-PEG _20K.

3. cell administration: take out sample solution and join in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 degree, CO ₂concentration 5% is cultivated after 24h, and 4 ℃, the centrifugal 5mim of 1400rpm, collects supernatant liquor and measure for ELISA.

4. result: as seen in Figure 9, after modifying through PEG, each group all has reduction in various degree to the hormesis of splenocyte, but contrasts with blank group, still can find out through after PEG modification, still plays the hormesis to splenocyte.Along with the increase of molecular weight, the expression level of IL-6 reduces.

the difference of the CpG serum stability that the PEG of embodiment 10, comparison different chain length modifies

1. preparation of samples: CpG and the CpG-PEG of the Nacl preparation same concentrations with 0.9% _5K, CpG-PEG _10K, CpG-PEG _20Ksolution.

2. administration: Balb/c mouse is divided into CpG group and CpG-PEG _5K, CpG-PEG _10K, CpG-PEG _20Kgroup, has taked mouse to carry out the administering mode of tail vein injection, every injected in mice 200 μ L CpG and CpG-PEG injection liquid.5min, 15min, 30min, 1h, 5h, 10h after injection respectively, 15h, 20h, nine time points of 25h are got blood by eye socket and are obtained blood sample.Owing to having volumetric wear in the process that blood treatment is become to serum, so each amount for taking blood should be not less than 50 μ L, avoid processing the serum amount obtaining and do not reach very little detection aequum.

3. sample preparation: the blood sample of obtaining is at room temperature left standstill to 30min, after blood fully solidifies, with the centrifugal 10min of speed of 3000rpm, take out supernatant liquor, and again with the centrifugal 10min of speed of 2200rpm.After centrifugal completing, take out supernatant liquor, be placed in-20 ℃ of freezing preservations.

4. result: adopt QRT-PCR to measure sample, experimental result shows, CpG-PEG _10K, CpG-PEG _20Karea under the drug-time curve be greater than CpG, CpG-PEG _5Kduring with CpG group medicine, curve does not have significant difference.CpG, CpG-PEG _5K, CpG-PEG _10K, CpG-PEG _20Ktransformation period be respectively 30min, 1h, 4h, 5h.Illustrate molecular weight be greater than 10K PEG modify CpG can improve CpG stability in vivo, avoid the degraded of nuclease,

embodiment 11, the interior immune effect difference of CpG body that relatively PEG of different chain length modifies

1. sample preparation, the CpG powder that the PEG of synthesized is modified, the effective CpG ODN(of every 100 μ g only counts CpG quality, disregards PEG quality) and be dissolved in 200 μ l PBS damping fluids, make the storing solution of 500 μ g/ml, now with the current.

2. immunization protocol, adopts the mode of abdominal injection to carry out administration.Below operation is aseptic technique.Every mouse is accepted 200 μ l dosage injections.When initial immunity, respectively the CpG of dosage CpG used and different chain length is added in ep pipe with liquid-transfering gun, then will add dosage chicken egg white used (OVA), finally adding PBS damping fluid to cumulative volume is 200 μ l.Within 7 days after mouse initial immunity, carry out, every mouse is accepted 200 μ l dosage injections, and initial immunity is shown in operation.Grouping situation sees the following form.

3. blood collection, after initial immunity the 14th day, gets respectively blood on the 21st day, and mouse blood collection is used the method for eye socket venous blood sampling.The blood sample of collecting is got supernatant by centrifugal (3000rpm, 10min), as still there being color, can repeated centrifugation process, and until completely without color.Be saved to-20 ℃ in order to analyzing.

4 ELISA detect mice serum antibody horizontal, come the anti-OVA IgG antibody of specificity in quantitative measurment serum, IgG1, the concentration level of IgG2a by the indirect ELISA that detects antibody.

5. statistical study, 5 of every group of experiment mices, in group, showing property difference is analyzed with one-side t check (one-tailed Student ' s test), and between group, for significant difference, single factor variance (one-way ANOVA) is analyzed, and data processing and mapping are used OriginPro8 to complete.In each figure, data are expressed as mean+SD (mean values ± standard deviation).

6. experimental result, for the result (Figure 10-13) of the 14th day, we can find out, for IgG, adjuvant group all showing property higher than OVA group (p<0.05), but each CpG-PEG group and the not difference of showing property (Figure 10) of common CpG ODN group; For IgG1, adjuvant group all showing property lower than without adjuvant group (p<0.05) (Figure 11); For IgG2a, showing property of adjuvant group is higher than OVA group (p<0.05), but CpG-PEG group and common CpG ODN group there is no the difference (Figure 12) of showing property; For disclosing, Th1 para-immunity is replied or the ratio of the IgG2a/IgG1 that Th2 para-immunity is replied, and each CpG-PEG group and not showing property difference of CpG group, illustrate that the immunostimulating effect of PEG-CpG is in the time of the 14th day, with common CpG group identical (Figure 13).Experimental result shows the group for each molecular weight PEG-CpG, IgG, and IgG1, all showing property difference not of tri-indexs of IgG2a, illustrates in the time of 14 days, PEG modifies the immune effect of CpG is not played to obvious impact.

At the 21st day, the specific immunity antibody expression of mouse was as figure below (14-17), and for the result of the 21st day, we can find out, for IgG, all showing property is higher than OVA group (p<0.05) (Figure 14) for adjuvant group; For IgG1, all showing property is lower than without adjuvant group (p<0.05) for adjuvant group, and each showing property of CpG-PEG group is lower than CpG group (Figure 15) simultaneously; For IgG2a, showing property of adjuvant group is higher than OVA group (p<0.05), but CpG-PEG group and common CpGODN group there is no the difference (Figure 16) of showing property; For disclosing, Th1 para-immunity is replied or the ratio of the IgG2a/IgG1 that Th2 para-immunity is replied, and each showing property of PEG-CpG group higher than CpG group (p<0.05) (Figure 17).Experimental result shows that the immunostimulating effect of CpG-PEG, in the time of the 21st day, is obviously better than common CpG group.

For the CpG-PEG group of each molecular weight, not showing property difference of tri-indexs of IgG, the IgG1 level of 20K molecular weight CpG-PEG is a little less than other two groups of CpG-PEG, and for disclosing, Th1 para-immunity is replied or the ratio of the IgG2a/IgG1 that Th2 para-immunity is replied simultaneously, CpG-PEG _20Kgroup, a little more than other two groups of CpG-PEG, illustrates in the time of 21 days, and 20K molecular weight PEG modifies the CpG that the immune effect of CpG is modified in lower molecular weight PEG the immuno-potentiation more showing.

the long-lasting difference of the CpG immune effect that embodiment 12, comparison different chain length PEG modify

1. sample preparation

The CpG powder that the PEG of synthesized is modified, the effective CpG ODN(of every 100 μ g only counts CpG quality, disregards PEG quality) be dissolved in 200 μ l PBS damping fluids, make the storing solution of 500 μ g/ml, now with the current.

2. immunization protocol

Adopt the mode of abdominal injection to carry out administration.Below operation is aseptic technique.Every mouse is accepted 200 μ l dosage injections.When initial immunity, the CpG of dosage CpG used and different chain length is added in eppendorf pipe with liquid-transfering gun, then will add dosage OVA used, finally adding PBS damping fluid to cumulative volume is 200 μ l.Within 7 days after mouse initial immunity, carry out, every mouse is accepted 200 μ l dosage injections, and initial immunity is shown in operation.Concrete immunization method sees the following form.

Group

Initial immunity (the 0th day)

Booster immunization (the 7th day)

?	OVA	Adjuvant	OVA	Adjuvant
					First group	10μg	Nothing	10μg	Nothing
Second group	10μg	CpG5μg	10μg	CpG5μg
					The 3rd group	10μg	CpG-PEG 5K5μg	10μg	CpG-PEG 5K5μg
The 4th group	10μg	CpG-PEG 10K5μg	10μg	CpG-PEG 10K5μg
					The 5th group	10μg	CpG-PEG 20K5μg	10μg	CpG-PEG 20K5μg

3. blood collection

After initial immunity, within the 14th, 21,28,35 days, get respectively blood, mouse blood collection is used the method for eye socket venous blood sampling.The blood sample of collecting is got supernatant by centrifugal (3000rmp, 10min), as still there being color, can repeated centrifugation process, and until completely without color.Be saved to-20 ℃ in order to analyzing.

4.ELISA detects mice serum antibody horizontal

We carry out the concentration level of specificity anti-OVA IgG antibody 2a in quantitative measurment serum and cytokine IFN-γ by detecting the indirect ELISA of antibody.

5. statistical study

5 of every group of experiment mices, in group, for showing property difference, one-side t check (one-tailed Student ' s test) is analyzed, and between group, significant difference for, single factor variance (one-way ANOVA) is analyzed, and data processing and mapping use OriginPro8 complete.In each figure, data are expressed as mean+SD (mean values ± standard deviation).

6. experimental result

From Figure 18 result, since 28 days, showing property of CpG-PEG group was higher than CpG group (p<0.05); 20K molecular weight PEG effect is better than 10k molecular weight PEG effect, is better than 5K molecular weight PEG effect.Experimental result shows that PEG molecular weight is larger, stronger, long-lasting better to the effect of prolongation CpG Half-life in vivo.In order to verify this result, we put mice serum IFN-γ concentration level and IgG2a concentration level to compare (Figure 19) together, find that both variation tendencies are basically identical.IgG2a is that humoral antibody is replied aspect performance Th1 para-immunity response effect, and IFN-γ is that cellullar immunologic response aspect shows Th1 para-immunity response effect.In our experiment, because both have similar variation pattern, illustrate and both can represent that body Th1 para-immunity replys degree, also illustrated that CpG ODN helps the Th1 para-immunity of protein antigen stimulation to reply, and not only comprises humoral immunoresponse(HI) but also comprise cellullar immunologic response.

embodiment 13, CpG-PEG and CpG are to breast cancer cell 4T1-pGL ₃ cytoactive experiment relatively

1. cell inoculation: 4T1-PGL ₃mouse source breast cancer cell is containing in 10% foetal calf serum RPMI-1640 substratum, in 37 ℃, and 5%CO ₂in incubator, cultivate.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, and cell is seeded in 96 orifice plates.

2. sample solution preparation: be mixed with CpG and the CpG-PEG solution that concentration is 25,100,250 μ g/ml, the filtering with microporous membrane degerming of 0.22 μ m with substratum.

3. cell administration: spend the night after cell is adherent on culture plate, take out original fluid, add respectively CpG and the CpG-PEG solution of 25,100, the 250 μ g/ml that use 1640 substratum of 200 μ l are mixed with.Using pure culture liquid as blank, 37 ℃, 5%CO ₂in incubator, cultivate respectively 24h and 72h.

4. detect: after having hatched, add 10 μ L CCK8 solution (note not generating bubble in hole again, they can affect the reading of OD value) to every hole.37 ℃, 5%CO ₂in incubator, hatch the suction brightness that is determined at 450nm place after 2h by microplate reader.

5. result: while cultivating 24h, the 4T1-pGL that the each concentration of CpG ODN1826 and CpG-PEG is intervened ₃cell, experimental group and the equal not statistically significant of blank comparing difference (P > 0.05).Cultivate 72h time point, CpG ODN and the CpG-PEG of 250 μ g/ml _20Kthe 4T1-pGL intervening ₃, the relatively blank group of the cytoactive right side reduces, but difference not statistically significant (p > 0.05) (seeing Figure 20).Show that CpG1826 and CpG-PEG are to 4T1-pGL ₃the growth unrestraint effect of cell.

embodiment 14, CpG-PEG experiment in vitro effect, investigate PEG and modify the immunostimulation impact of CpG on BMDC

The collection of 1.mDC cell and cultivation: get 6 of C57/BL6 mouse, male, disconnected cervical vertebra is put to death, and soaks approximately 15 minutes in 75% ethanol; Mouse is moved in Bechtop, separate shin bone and femur, remove muscle and reticular tissue, cut off epiphysis end, draw serum-free without dual anti-RPMI1640 nutrient solution with syringe, medullary cell is gone out to bone look and turned white; Collect medullary cell, the centrifugal 5min of 1400rpm, abandons supernatant, add 7ml Tris ?NH ₄cl solution splitting erythrocyte, is placed under room temperature and leaves standstill 5 minutes; Centrifugal 5 minutes of 4 ℃ of 1400rpm, abandon supernatant, add the RPMI-1640 Eddy diffusion dispersion of 24ml containing the granulocyte-macrophage colony stimutaing factor of 10ng/ml mouse restructuring (mGM ?CSF) and 1ng/ml interleukin-4 (IL-4), add in 66 porocyte culture plates, every hole 4ml, 37 ℃, 5%CO ₂cultivate; After 72 hours, remove all cells nutrient solution and suspension cell, only retain attached cell, then every hole add 4ml containing 10ng/ml mGM ?CSF and 1ng/ml IL ?4 RPMI-1640, continue to cultivate; Within the 5th day, 2ml nutrient solution is shifted out in every hole, then add into 2ml containing 10ng/ml mGM ?CSF and 1ng/ml IL ?4 RPMI-1640, continue to cultivate; The 6th day time, collect suspension cell and slight attached cell, be the BMDC in mouse source, can be used for next step experiment.The mDC collecting, as in 96 orifice plates, is hatched IFN-γ (5ng/ml) and BMDC after 12h, remove substratum, add 1640 substratum, in cell culture incubator, hatch 2h.

2. the preparation of sample solution: the CpG and the CpG-PEG that are respectively 5,2.5,1.25nmol/ml with PBS compound concentration _20Ksolution.

3. cell administration: take out the sample solution 5 μ l of different concns, join in 96 orifice plates, three multiple holes of each sample.In constant temperature cell culture incubator, 37 degree, CO ₂concentration 5% was cultivated after 24 hours, and 4 ℃, the centrifugal 5mim of 1400rpm, collecting cell supernatant liquor, ELISA measures the expression amount of IL-p70.

4. result: can find out by upper result, after modifying through PEG, each group all has reduction in various degree to the hormesis of splenocyte, but contrast with blank group, still can find out through after PEG modification, still play the hormesis of BMDC, IL-12p70 secretion is enhanced, and splenocyte is stimulated to secretion IL-6's have a similarity (Figure 21).

the action effect of CpG after embodiment 15, PEG modify to regulatory T cells

1. sample preparation

The CpG powder that the PEG of synthesized is modified, the effective CpG ODN of every 100 μ g is dissolved in 200 μ l PBS damping fluids, makes storing solution, now with the current.

2. immunization protocol

8 weeks large C57 mouse, adopt the mode of abdominal injection to carry out administration.Below operation is aseptic technique.Every mouse is accepted 200 μ l dosage injections.Dosage CpG used and CpG-PEG are added in ep pipe with liquid-transfering gun, then will add dosage OVA used, finally adding PBS damping fluid to cumulative volume is 200 μ l.After one week, then give OVA+CpG/CpG-PEG, after one week, again give OVA, put to death, get spleen and lymphoglandula.

3. from spleen and lymphoglandula, isolate T cell, turn out T cell, with OVA stimulation, measure IL-10 expression level.

4. the IL-10 expression level of result: CpG-PEG group, lower than CpG group, proves that CpG-PEG can reduce the action effect of CpG for regulatory T cells (Treg).

can be on the impact of normal mouse inherent immunity after the CpG intravenous administration that embodiment 16, PEG modify

1. sample preparation

The CpG powder that the PEG of synthesized is modified, the effective CpG ODN of every 100 μ g is dissolved in 200 μ l PBS damping fluids, makes storing solution, now with the current.

2. immunization protocol

8 weeks large C57 mouse, adopt intravenous mode to carry out administration, mouse are divided into 3 groups at random, 5 every group.1. CpG1826-iv: tail vein injection CpG-1826, twice weekly; 2. CpG-PEG _20k– iv: intravenous injection CpG-PEG, twice weekly; 3. control group-iv: intravenous injection 0.9%NaCl, twice weekly.Below operation is aseptic technique.Get weekly blood once, totally two weeks.The blood sample of collecting is got supernatant and is added in an other clean ep pipe by centrifugal (3000rmp, 10min), as still there being color, can repeated centrifugation process, and until completely without color.Be saved to-20 ℃ for elisa assay.

3. result: can find out from experimental result, the expression level of the IL-12p70 of CpG-PEG group improves with respect to CpG group, proves that CpG-PEG strengthens mouse innate immune response.

immune antitumous effect in the body of CpG after embodiment 17, PEG modify

1. model is set up and grouping: BALB/c mouse breast cancer cell line 4T1-PGL3 is incubated at containing 10%FBS, in 1% dual anti-RPMI1640 substratum, and 37 ℃, 5%CO ₂, in the time that monolayer cell lamella reaches 90% fusion, 0.25% trypsin digestion and cell goes down to posterity.The 4T1-pGL3 cell of collecting logarithmic phase, PBS washes 1 time, cell counting.The upper left side mammary subcutaneous of BALB/c mouse is with 5*10 ⁵cell/only plant knurl.Treat that tumour grows to diameter and has 5mm left and right to start administration.There is the mouse of tumour to be divided at random 5 groups, 5 every group by long.1. CpG1826-iv: the CpG-1826 of tail vein injection 200ul (4nmol), twice weekly; 2. CpG-PEG _20k– iv: the CpG-PEG20K of intravenous injection 200 μ l, twice weekly; 3. control group-iv: the 0.9%NaCl of intravenous injection 200 μ l, twice weekly; 4. CpG-1826-pt group: swollen tumor-side injection 200 μ l(4nmol) CpG-1826, twice weekly; 5. control group-pt group: the 0.9%NaCl of swollen tumor-side injection 200 μ l, twice weekly.

2. tumor growth comparison

Observe tumor propagation situation, use every three days the long * of a gross tumor volume of vernier caliper measurement (V=) 1/2* wide ².After one month, finish to observe.

3. in tumor-bearing mice, in serum, the expression amount of IL-12p70 is measured

Get weekly blood once, altogether surrounding.The blood sample of collecting is got supernatant and is added in an other clean ep pipe by centrifugal (3000rmp, 10min), as still there being color, can repeated centrifugation process, and until completely without color.Be saved to-20 ℃ for elisa assay.

4. result:

Can find out CpG-pt group and CpG-PEG from tumor growth curve _20Khave showing property difference (P<0.05) with 0.9%NaCl group, CpG-iv group and not showing property difference (P>0.05) of 0.9%Nacl, show that the CpG antitumous effect after PEG modifies strengthens (seeing Figure 22).In the mice serum of CpG-PEG group, the level of IL-12p70 is apparently higher than other groups (P<0.05), there is not showing property difference (P>0.05) in CpG-iv and CpG-pt group, shows stronger (the seeing Figure 23) of effect of the CpG immune stimulatory emiocytosis IL-12p70 after PEG modifies.

Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims (10)

1. the CpG oligonucleotide that PEG modifies.

2. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that mw > >=10K of described PEG.

3. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that, the skeleton of described oligonucleotide is full thio-modification, part thio-modification or without thio-modification.

4. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that, the site that described PEG modifies is 5 '-end or 3 '-end.

5. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that, described structure is shown in formula 1 or formula 2, the CpG unit that described OLIGO oligonucleotide wherein comprises 1-5 unit;

6. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that, the sequence of described CpG oligonucleotide is:

The CpG-7 of sequence as shown in SEQ ID NO.1,

The CpG-8 of sequence as shown in SEQ ID NO.2,

The CpG-13 of sequence as shown in SEQ ID NO.3,

The CpG1826 of sequence as shown in SEQ ID NO.4,

The CpG7909 of sequence as shown in SEQ ID NO.5,

The CpG-H7 of sequence as shown in SEQ ID NO.6,

The CpG-H8 of sequence as shown in SEQ ID NO.7,

The CpG-H13 of sequence as shown in SEQ ID NO.8,

The CpG-H1826 of sequence as shown in SEQ ID NO.9.

7. the CpG oligonucleotide that PEG as claimed in claim 1 modifies, is characterized in that, the CpG oligonucleotide that described PEG modifies can be synthesized into by the method comprising the steps;

Route a, joins the CpG of sulfydryl modification in Tris-HCL, blows N ₂5min, then add three (2-carbonyl ethyl) microcosmic salt hydrochlorate, mix, then add mPEG _20K-Mal, N in whole process ₂protection, vortex mixes, and 25 ℃, 500-1300rpm reacts 5h; By the mixture dichloromethane extraction of the product obtaining, collect dichloromethane layer solution, repeatedly extract three times, volatilize with nitrogen, add ultrapure water to dissolve; 4 ℃ of dialysed overnight of dialysis tubing of finally obtained synthetic product being made with the dialysis membrane of 3500Da, dialysis finishes sample freeze-drying, to obtain final product;

Route b, the mPEG that alkynyl-modified CpG and nitrine are modified, in the reaction solvent of being prepared for 1:1 by volume by water and methyl alcohol, does at cupric sulfate pentahydrate and sodium ascorbate under the condition of catalyzer and vibrates and mix, and reacts 18h and get final product with 37 ℃;

8. the CpG oligonucleotide of a PEG modification as claimed in claim 1 improves the application in the IL-12 expression amount medicine in serum in preparation.

9. the CpG oligonucleotide of a PEG modification as claimed in claim 1 is induced for endogenous antigen as the application in the immunogenicity medicine of tumour antigen in preparation.

10. the application of the CpG oligonucleotide of a PEG modification as claimed in claim 1 in the immunocompetence drug of preparation stimulation B cell, plasmacytoid dendritic cells, medullary system dendritic cell, scavenger cell, peripheral mononuclear cells.

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