CN1296378C - CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof - Google Patents

Abstract

The invention discloses an immunostimulatory oligonucleotide with high immune activity and the gene sequence of the oligonucleotide antagonized to the immunostimulatory oligonucleotide, and discloses that the immunostimulatory oligonucleotide with high immune activity is prepared The application in the treatment of infectious diseases, allergic diseases, immunodeficiency diseases and tumor drugs; the application of oligonucleotides antagonizing immune stimulating oligonucleotides in the preparation of drugs for treating SIRS and sepsis.

Description

Gene order and the application thereof of the CpG-N ODN of the CpG-S ODN of high immunological activity and antagonism CpG-S ODN effect

Technical field

The present invention relates to a kind of oligonucleotide and, specifically, relate to a kind of immunostimulatory oligonucleotide (CpG-S ODN) and it is had the oligonucleotide (CpG-N ODN) of antagonistic action in pharmaceutically application.

Background technology

Containing not, the oligonucleotide fragment of methylated CpG is the least action unit of DNA of bacteria, therefore what have immunostimulation contains not that the oligonucleotide of methylated CpG is known as immunostimulatory oligonucleotide (Immnostimulatory oligonucleotides, CpG-S ODN).Its constitutional features is to contain i.e. six aggressiveness of 6 base complementrity palindromes of CpG primitive, must contain a CpG dinucleotides at least in this six aggressiveness, 5 ' the end of CpG must have a purine, similar ApA, GpA or GpT etc., 3 ' end must have two pyrimidines such as TpT, and known six aggressiveness with immunostimulation comprise AACGTT, AGCGCT etc. at present.

CpG-S ODN has the effect of two aspects: low dose of CpG-S ODN has immunocompetence widely to the mouse immune cell, heavy dose then can be induced systemic inflammatory response syndrome, and (Systemic inflammatoryresponse syndrome is SIRS) with pyemic generation.

Studies show that in a large number: 1. low dose of CpG-S ODN can directly or indirectly activate dendritic cell, B cell, monokaryon-scavenger cell, NK cell and cytotoxic T cell (CTL); Induce Th1 cytokines releases such as IFN-γ.Therefore, use can effectively suppress the mouse tumor growth, promote tumor regression separately, and can form the protective immunity memory, thereby can effectively prevent tumor recurrence.In addition,, therefore also can treat malignant tumour, have a good application prospect aspect tumor prevention and the treatment with the tumor vaccine compatibility because CpG ODN is a kind of good Th1 type vaccine adjuvant.Owing to still lack the CpG-S ODN that people's immunocyte is had high immunological activity at present, therefore need the CpG-S ODN of searching high immunological activity significant with treatment to tumor prevention.2. CpG-S ODN induces the Th1 cytokines to discharge and suppresses Th2 type para-immunity and reply, so can be used to treat anaphylactic disease such as the asthma that Th2 type para-immunity is replied mediation.3. CpG-S ODN can activate cells such as dendritic cell, B cell, monokaryon-scavenger cell, therefore treats infectious diseases, the immunologic hypofunction disease that pathogenic agent such as bacterium or virus cause.

Because CpG-S ODN extensively is present in the DNA of bacteria, when infectation of bacteria took place, a large amount of breedings of bacterium can cause CpG-S ODN massive duplication.Excessive CpG-S ODN discharges the various kinds of cell factors such as TNF-α, IL-1, IL-6 and IL-12 in a large number by activated mononuclear-phagocytic cell system and causes SIRS and pyemic generation, and its case fatality rate does not still have effective treatment means so far up to 50-80%.Have and contain unmethylated CpG primitive among the reported literature bacterial genomes DNA and not necessarily have immunostimulation, even have the blocking-up or in and the CpG-SODN effect, this class oligonucleotide is called as CpG-N ODN (CpG-neutralizing oligonucleotides, CpG-N ODN), therefore research is significant to SIRS and pyemic treatment to the CpG-N ODN that CpG-S ODN has antagonistic action.

Summary of the invention

The object of the present invention is to provide a kind of immunostimulatory oligonucleotide, it has high immunological activity, can form the protective immunity memory, can suppress Th2 type para-immunity and reply.

Another object of the present invention is to provide a kind of application of immunostimulatory oligonucleotide in prevention and treatment infectious diseases, anaphylactic disease, immunodeficiency diseases and tumour medicine of high immunological activity.

A further object of the present invention is to provide a kind of oligonucleotide to the immunostimulatory oligonucleotide antagonism (CpG-NODN), have blocking-up or in and the effect of CpG-S ODN.

An also purpose of the present invention is to provide a kind of oligonucleotide to the immunostimulatory oligonucleotide antagonism being used for preparing the application for the treatment of SIRS and medication for treating pyemia.

In order to realize the object of the invention, a kind of immunostimulatory oligonucleotide has following gene order:

GGC?GGC?GGC?GCG；(1)

CGC?GGC?GCC?GGG；(2)

GGC?GGC?GGC?GGA；(3)

CCG?CGC?GCG?GGC；(4)

TGG?CGC?GGG?GCG；(5)

GCC?GGC?GCC?CGG；(6)

GCG?CGC?GCG?GTA；(7)

CCA?GGC?GGC?GGG；(8)

GGC?CGC?GGG?GGT；(9)

CAG?CCC?GGG?GGA；(10)

GCT?CCC?GGG?CTT；(11)

GCG?CTC?GGG?CTT；(12)

CCG CCC GCG CCT; (13) or

Wherein a kind of of GCA CCC GCG CGC (14).

Wherein, preferred gene order:

GGC?GGC?GGC?GCG；

CCG?CGC?GCG?GGC；

TGG?CGC?GGG?GCG；

GCC?GGC?GCC?CGG；

GGC?CGC?GGG?GGT；

CAG?CCC?GGG?GGA；

GCT?CCC?GGG?CTT；

GCG CTC GGG CTT; Or

Wherein a kind of of CCG CCC GCG CCT.

The immunostimulatory oligonucleotide of a kind of high immunological activity of the present invention has high immunological activity; can form the protective immunity memory; can suppress Th2 type para-immunity and reply, it is used for preparing the application of treatment infectious diseases, anaphylactic disease, immunodeficiency diseases and tumour medicine.

In order to realize another goal of the invention, a kind of oligonucleotide to the immunostimulatory oligonucleotide antagonism has following gene order:

GGC?CGC?GGG?GAC；(15)

GCC?GCC?GCC?GCC；(16)

CGC?CTC?GGG?GAG；(17)

TGC?CGC?GGC?AGA；(18)

CTC?CAC?GTG?CCG；(19)

GCC GCC GCC GTC; (20) or

Wherein a kind of of GCT GCC GCC GCC (21).

Its preferred gene order:

CGC?CTC?GGG?GAG；

TGC?CGC?GGC?AGA；

CTC?CAC?GTG?CCG；

GCC GCC GCC GTC; Or

Wherein a kind of of GCT GCC GCC GCC.

A kind of oligonucleotide of the present invention to the immunostimulatory oligonucleotide antagonism have blocking-up or in and CpG-S ODN induce hPBMC to discharge the ability of TNF-α, it is in the application that is being used for preparing treatment SIRS and medication for treating pyemia.

In order to obtain highly active CpG-S ODN and CpG-N ODN, researchist of the present invention starts with from the genome of virus and carries out.

Those skilled in the art understand all small viruses and there is numerous not methylated CpG dinucleotides in reverse transcription virus gene group DNA, and wherein many have a CpG-S ODN constitutional features.Therefore infer that in theory it should have very strong immunostimulation.But true on the contrary, viral DNA not only can be escaped immunity identification and the removing of body immune system to it, and can duplicate in host cell, and this ability shows more significantly in retrovirus.Therefore should exist in the viral DNA blocking-up or in and the oligonucleotide sequence of CpG-S ODN effect, but and do not know what its constitutional features is?

The research of relevant adenovirus DNA (Adenoviral DNA, Adv DNA) has given the enlightenment that the researchist is highly profitable.Although all contain the suitable not methylated CpG dinucleotides of quantity among serotype 2,5Adv DNA (subgenus C) and the serotype 12Adv DNA (subgenus A), therefore infer that they all should have identical immunostimulation.DNA has very strong immunostimulation but the experimental result of Krieg etc. shows serotype 12Adv, and serotype 2,5Adv DNA then not only do not have immunostimulation, and has blocking-up CpG-S ODN and induce TNF-α, the effect of IL-6 excretory.Researchist of the present invention has also repeated this experiment, and the result is consistent with Krieg's.This presentation of results: have CpG-S ODN among (1) serotype 12Adv DNA, exist among serotype 2, the 5 Adv DNA have blocking-up or in and the oligonucleotide sequence of EC DNA or CpG-S ODN effect; (2) the CpG dinucleotides participates in the composition of CpG primitive and brings into play immunostimulation among the serotype 12Adv DNA; (3) the CpG dinucleotides may not participate in the composition of CpG-S ODN fully and brings into play immunostimulation among serotype 2, the 5Adv DNA, because serotype 2,5Adv dna sequence analysis are shown, the CpG that participates in CpG primitive composition only accounts for the 1/15-30 of CpG total quantity, points out the effect of these CpG primitives to be suppressed by other oligonucleotide with neutralizing effect.

According to above analysis, researchist of the present invention infers that serotype 12Adv DNA contains the CpG-S ODN with very strong immunostimulation, and exist in the serotype 2,5Adv DNA natural have blocking-up or in and the CpG-N ODN of CpG-S ODN, the sequence signature of CpG-N ODN may change relevant with CpG two ends oligonucleotide sequence characteristics.

Therefore, researchist of the present invention adopts bioinformatics technique, with the CpG dinucleotides is " electronic probe " (the CpG dinucleotides is retrieval " label "), with six aggressiveness among the CpG-S ODN is the comparison template, serotype 2,5 and 12Adv DNA, EC DNA are carried out sequential analysis, seek the oligonucleotide sequence that has 12 bases of common structure feature among serotype 2, the 5Adv DNA; And by after the active screening of external biological, obtain having blocking-up or in and the oligonucleotide (CpG-N ODN) of CpG-S ODN effect; Observe the provide protection of CpG-NODN, the effect of clear and definite CpG-N ODN in the SIRS control to DNA of bacteria mediation SIRS animal model mouse.

Serotype 2,5 and 12Adv DNA, EC dna sequence dna (ACCESSION:NC_001405, M73260, NC_001460, NC_000913) have been obtained the Nucleotide database of researchist of the present invention from PubMed.

Adopt bioinformatics technique, use biosoftwares such as Primer Premier 5, BioEdit version 5.0.6,256 kinds of Hexanucleotide sequences that contain CpG among serotype 2,5 and 12Adv DNA, the EC DNA are analyzed and compared, determined distinctive 19 kinds of Hexanucleotide sequences that contain CpG among serotype 2, the 5Adv DNA; Use BioEdit version 5.0.6, finding out among serotype 2, the 5Adv DNA with these 19 kinds of CpG Hexanucleotides is 12 base oligonucleotide (ODN) of core; Use biosoftware RNAstructure version 3.71, the secondary structure of 12 base ODN is predicted, found out 4 kinds of possible common structure features: (1) c-g is positioned at and does the ring top; (2) g-c is positioned at and does the ring top; (3) c-g or g-c are positioned at and do the ring side; (4) no dried ring structure (see figure 1), thus first design finished to CpG-N ODN molecule.

Synthesized the ODN of 10 phosphoric acid sulfo-skeletons according to this design, wherein c-g is positioned at and does ring vertical 3 (ODN1-3), g-c and be positioned at and do ring vertical 3 (ODN 4-6), c-g or g-c and be positioned at 2 (ODN9-10) that do ring lateral 2 (ODN7-8), no dried ring structure formation.Researchist of the present invention identifies the extracorporeal biology activity of 10 ODN, finds the ODN of 2 no obvious irritation, and 8 ODN have the ability of inducing hPBMC to discharge TNF-α, and promptly 8 ODN are immunostimulatory oligonucleotide (CpG-S ODN).

Use biosoftware RNAstructure version 3.71, secondary structure free energy to ODN is analyzed, on this basis, pungency and the relation between the structure to 10 ODN have carried out analyzing and finding, the biological activity of ODN may be irrelevant with 4 kinds of common structure features of prediction secondary structure; And may be closely related with the free energy height of prediction secondary structure.Therefore, use 3.71 pairs of CpG-NODN molecules of biosoftware RNAstructure version to carry out design again.Synthesized the ODN of 11 phosphoric acid sulfo-skeletons according to this design, researchist of the present invention identifies the extracorporeal biology activity of 11 ODN, finds the ODN of 5 no obvious irritation, and 6 ODN are immunostimulatory oligonucleotide (CpG-S ODN).

Researchist of the present invention obtains 14 immunostimulatory oligonucleotides (CpG-SODN) altogether in twice screening experiment, its gene order is: GGC GGC GGC GCG; CGC GGC GCC GGG; GGC GGC GGCGGA; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GCG CGCGCG GTA; CCA GGC GGC GGG; GGC CGC GGG GGT; CAG CCC GGG GGA; GCT CCC GGG CTT; GCG CTC GGG CTT; CCG CCC GCG CCT; Or wherein a kind of of GCA CCC GCGCGC; Wherein preferably: GGC GGC GGC GCG; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GGC CGC GGG GGT; CAG CCC GGGGGA; GCT CCC GGG CTT; GCG CTC GGG CTT; Or wherein a kind of of CCG CCC GCG CCT.

Researchist of the present invention identifies the activity of the ODN inhibition CpG-S ODN of 7 no obvious irritation simultaneously, show that these 7 ODN all have the ability that CpG-S ODN inductive hPBMC discharges TNF-α that suppresses, promptly 7 ODN are the oligonucleotide (CpG-N ODN) to the immunostimulatory oligonucleotide antagonism, and its gene order is: GGC CGC GGG GAC; GCC GCC GCC GCC; CGC CTC GGG GAG; TGCCGC GGC AGA; CTC CAC GTG CCG; GCC GCC GCC GTC; Or wherein a kind of of GCT GCC GCCGCC; Preferably: CGC CTC GGG GAG; TGC CGC GGC AGA; CTC CACGTG CCG; GCC GCC GCC GTC; Or wherein a kind of of GCT GCC GCC GCC.

The oligonucleotide (CpG-N ODN) that immunostimulatory oligonucleotide of the present invention (CpG-S ODN) reaches the immunostimulatory oligonucleotide antagonism can adopt this area synthetic method commonly used synthetic, such as the solid phase phosphoramidite triester method.

Researchist of the present invention carries out the pharmacology analysis and research through and synthetic Nucleotide designed to the present invention, find that immunostimulatory oligonucleotide of the present invention (CpG-S ODN) has immunocompetence widely, directly or indirectly activates dendritic cell, B cell, monokaryon-scavenger cell, NK cell and cytotoxic T cell (CTL); Induce Th1 cytokines releases such as IFN-γ, can form the protective immunity memory, can suppress Th2 type para-immunity to reply, it is used for preparing the application for the treatment of infectious diseases, anaphylactic disease such as asthma, immunodeficiency diseases and tumour medicine.

Antagonism oligonucleotide of the present invention (CpG-N ODN) have blocking-up or in and CpG-S ODN induce hPBMC to discharge the ability of TNF-α, it is in the application that is being used for preparing treatment SIRS and medication for treating pyemia.

Description of drawings

4 kinds of possible common structure feature: c-g of secondary structure of Fig. 1 12 base ODN prediction are positioned at and do the ring top

4 kinds of possible common structure feature: g-c of secondary structure of Fig. 2 12 base ODN prediction are positioned at and do the ring top

4 kinds of possible common structure features of secondary structure of Fig. 3 12 base ODN prediction: c-g or g-c are positioned at and do the ring side

4 kinds of possible common structure features of secondary structure of Fig. 4 12 base ODN prediction: do not have dried ring structure

Embodiment

Following embodiment is for further describing the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.

Experimental example 1

This experimental example is to obtain CpG-S ODN and CpG-N ODN gene order by adopting bioinformatics technique.

1. serotype 2,5 and 12Adv DNA, ECDNA sequence (ACCESSION:NC_001405, M73260, NC_001460, NC_000913) have been obtained the Nucleotide database from PubMed.

2. employing bioinformatics technique, use biosoftwares such as Primer Premier 5, BioEdit version 5.0.6,256 kinds of Hexanucleotide sequences that contain CpG among serotype 2,5 and 12Adv DNA, the EC DNA have been carried out analyzing and comparing (seeing Table 1), according to: 1. the frequency of occurrences is higher in serotype 2,5Adv DNA; 2. the frequency of occurrences is lower in Adv 12DNA and EC DNA; 3. 3 principles such as Adv2:Adv12 〉=2 or Adv2:EC 〉=5 have been determined distinctive 19 kinds of Hexanucleotide sequences that contain CpG among serotype 2, the 5AdvDNA.

256 kinds of Hexanucleotide sequential analysis and comparisons that contain CpG among table 1 Adv 2,5,12DNA and the EC DNA

Sequence number	Sequence	Adv?2 ?		Adv?5 ?		Adv?12 ?		EC?DNA ?		? Adv2:Adv12	? Adv2:EC
												Frequency	Frequency	Frequency	Frequency	Frequency	Frequency	Frequency	Frequency
		1 2 3	aacgaa aacgag aacgac	5 10 10	2.06E-03 4.12E-03 4.12E-03	8 10 8	3.31E-03 4.14E-03 3.31E-03	6 8 4	4.00E-03 5.33E-03 2.67E-03											1246 589 1136	3.59E-03 1.70E-03 3.28E-03	0.51 0.77 1.54	0.57 2.42 1.26
……
												254 255 256	ttcgtg ttcgtc ttcgtt	2 4 4	8.24E-04 1.65E-03 1.65E-03	1 3 4	4.14E-04 1.24E-03 1.66E-03	4 8 3	2.67E-03 5.33E-03 2.00E-03	848 1296 1321	2.45E-03 3.74E-03 3.81E-03	0.31 0.31 0.82	0.34 0.44 0.43
Amount to theoretical mean		2428 9.48	1.00 3.91E-03	2414 9.43	1.00 3.91E-03	1500 5.86	1.00 3.91E-03	346636 1354.05	1.00 3.91E-03

3. use BioEdit version 5.0.6, finding out among serotype 2, the 5Adv DNA with these 19 kinds of CpG Hexanucleotides is 12 base oligonucleotide (ODN) of core; (this software is the secondary structure of measurable RNA both to use biosoftware RNAstructure version 3.71, the also secondary structure of measurable DNA), secondary structure to 12 base ODN is predicted, has found out 4 kinds of possible common structure features: (1) common trait 1:c-g is positioned at and does the ring top; (2) common trait 2:g-c is positioned at and does the ring top; (3) common trait 3:c-g or g-c are positioned at and do the ring side; (4) common trait 4: do not have dried ring structure (see figure 1).

4. the 12 base ODN that synthesized 10 phosphoric acid sulfo-skeletons, wherein c-g is positioned at and does ring vertical 3 (ODN1-3), g-c and be positioned at and do ring vertical 3 (ODN 4-6), c-g or g-c and be positioned at 2 (ODN9-10) that do ring lateral 2 (ODN7-8), no dried ring structure formation.

5. the active evaluation work of the extracorporeal biology of 10 ODN: separate and cultivator peripheral blood mononuclear cell (hPBMC), adjusting the hPBMC concentration of cell suspension is 1 * 10 ⁶/ ml adds in 24 orifice plates, every hole 1ml.Put 37 ℃ of CO ₂Incubator adds 10 μ g/ml ODN respectively to be screened after cultivating 2h, puts 37 ℃ of CO ₂Incubator is got cells and supernatant after cultivating 4h, adopt the double-antibody sandwich elisa method to measure the concentration of TNF-in the cells and supernatant, clear and definite 10 ODN induce the ability (seeing Table 2) of hPBMC release TNF-α, have filtered out the ODN:ODN1 and the ODN 6 of 2 no obvious irritation in view of the above; And other 8 ODN have the ability of inducing hPBMC to discharge TNF-α.

Table 2.10 oligonucleotide 10 μ g/ml induce hPBMC to discharge mensuration (n=3, the x ± s) of TNF-α ability when stimulating

Sequence number	Sequence	TNF-α(pg/ml)
			Blank ODN1 ODN2 ODN3 ODN4 ODN5 ? ODN6 ODN7 ODN8 ODN9 ODN10	- GGC?CGC?GGG?GAC GGC?GGC?GGC?GCG CGC?GGC?GCC?GGG GGC?GGC?GGC?GGA CCG?CGC?GCG?GGC GCC?GCC?GCC?GCC TGG?CGC?GGG?GCG GCC?GGC?GCC?CGG GCG?CGC?GCG?GTA CCA?GGC?GGC?GGG	52±4 92±4 2034±414 579±97 871±150 1090±207 285±15 2592±66 1198±386 674±171 773±105

6. use biosoftware RNAstructure version 3.71, secondary structure free energy to ODN is analyzed, on this basis, pungency and the relation between the structure to above-mentioned 10 ODN have carried out analyzing and finding, the biological activity of ODN may be irrelevant with 4 kinds of common structure features of prediction secondary structure; And may be closely related with the free energy height of prediction secondary structure: do not contain GGCG structure and free energy and may be the ODN (seeing Table 3) of no immunostimulating greater than-1.0 ODN.Therefore, use 3.71 couples of CpG-N ODN of biosoftware RNAstructure version molecule to carry out design again, synthesized the ODN of 11 phosphoric acid sulfo-skeletons according to this design.

The pungency of 10 ODN of table 3. and the relation between the structure

? ODN ?	Sequence	Free energy (kcal/mol)	The common structure feature	TNF-α ? (pg/ml)
					1 2 3 4 5 6 7 8 9 10	GGC?CGC?GGG?GAC GGC?GGC?GGC?GCG CGC?GGC?GCC?GGG GGC?GGC?GGC?GGA CCG?CGC?GCG?GGC GCC?GCC?GCC?GCC TGG?CGC?GGG?GCG GCC?GGC?GCC?CGG GCG?CGC?GCG?GTA CCA?GGC?GGC?GGG	-0.4 -3.9 -0.1 -1.6 -1.0 -0.2 -1.7 -3.1 ? ?	Common structure feature 1 common structure feature 1 common structure feature 1 common structure feature 2 common structure features 2 common structure features 2 common structure features 3 common structure features 3 common structure features 4 common structure features 4	92±4 2034±414 579±97 871±150 1090±207 285±15 2592±66 1198±386 674±171 773±105

7. the active evaluation work of the extracorporeal biology of 11 ODN: method has filtered out the ODN:ODN15,18,19,20,21 of 5 no obvious irritation in view of the above with step 5; And other 6 ODN have the ability (seeing Table 4) of inducing hPBMC to discharge TNF-α.

11 oligonucleotide of table 4., 10 μ g/ml induce hPBMC to discharge mensuration (n=3, the x ± s) of TNF-α ability when stimulating

Sequence number	Sequence	TNF-α(pg/ml)
			Blank ODN11 ODN12 ODN13 ODN14 ODN15 ODN16 ODN17 ? ODN18? ODN19? ODN20? ODN21	- GGC?CGC?GGG?GGT CAG?CCC?GGG?GGA GCT?CCC?GGG?CTT GCG?CTC?GGG?CTT CGC?CTC?GGG?GAG CCG?CCC?GCG?CCT GCA?CCC?GCG?CGC TGC?CGC?GG?CAGA CTC?CAC?GTG?CCG GCC?GCC?GCC?GTC GCT?GCC?GCC?GCC	36±2 2979±7 2485±35 2153±166 1867±98 200±130 1842±133 676±135 156±17 134±10 241±99 251±43

Experimental example 2

This experimental example is to study the synthetic method of oligonucleotide.

Method of the present invention adopts the solid phase phosphoramidite triester method, briefly, is that the monomer in the solution forms 3 ' → 5 ' phosphodiester bond by condensation reaction, thereby is connected on the solid support.

1. basic material:

(1) upholder: solid phase synthesis is fixed on nucleic acid and finishes building-up reactions on the solid phase carrier, the most frequently used solid phase carrier is controlled micropore glass pearl (CPG, controlled pore glass), decide according to the length of institute's synthetic oligonucleotide in the aperture of CPG, when generally synthesizing chain length less than 60nt, selects aperture 500 dusts, chain length is during greater than 60nt, use 1000 dust CPG, the condensation efficiency that uses CPG can satisfy the synthetic condition that reaches the oligonucleotide of 175nt up to 98%-99.9%.CPG is by connecting the hydroxyl covalent attachment of compound and incipient nucleus thuja acid, and 5 ' hydroxyl of Nucleotide is protected with dimethoxytrityl (DMT).

(2) monomer: synthetic used monomer is a nucleoside phosphoramidites, is the Nucleotide through chemically modified, contains following several functional group.

1. 3 ' P goes up diisopropylamino, the functional group that condensation is used

2. 3 ' P goes up the nitrile ethyl, and protecting group is sloughed behind synthetic the finishing.

3. 5 '-DMT, protecting group is sloughed before the condensation.

4. the phenylformic acid protecting group on the heterocyclic amino group of A and C is sloughed behind synthetic the finishing.

5. G goes up the different propionyl protecting group on the purine skeleton amino, sloughs behind synthetic the finishing.

(3) reactions steps:

The synthetic the first step----deblocking (Deblocking) is removed CPG with trichoroacetic acid(TCA) and is connected DMT on the nucleosides, to expose 5 ' hydroxyl, next step condensation of confession.This step should be noted that TCA is the acid of the last one, may have depurination, so TCA and oligonucleotide do not exceed schedule time duration of contact.

Second the step----activation (Activation), before condensation, monomer and tetrazolium are mixed and fed into synthetic post, this moment, tetrazolium provided the N atom of a proton to diisopropylamino on the 3 ' phosphoric acid, protonated Diisopropylamine is a good free group, forms this active intermediate of phosphoramidite tetrazolium with tetrazolium.This step tetrazolium is excessive to have guaranteed the monomer activation fully.

The 3rd step----connected (Coupling), when the Nucleotide that phosphoramidite tetrazolium and CPG are connected collides, with its 5 ' hydroxyl generation nucleophilic reaction, condensation takes place also take off tetrazolium, one of synthetic oligonucleotide chain extension.This step monomer connects the excessive high-level efficiency that connects that guaranteed of 5 '-hydroxyl on the Nucleotide with respect to CPG.

The 4th step----block (Capping) is extended in circulation subsequently in order to prevent the unreacted 5 ' hydroxyl that links to each other with CPG, need make it sealing after ligation is fully carried out, and this hydroxyl is sealed in acetylize commonly used.Face with very strong acetylation reagents of formation one activity such as preceding mixing diacetyl oxide and N-Methylimidazoles, be condensed into ester bond with a small amount of 5 hydroxyls for the participation ligation.Because the few and active height of acetylation reagent and fully excessive of hydroxyl that must seal, this step speed of response is very fast, and several seconds are promptly enough.The oversize block time have take place in unexpected position acetylization reaction may, and increase the danger that diacetyl oxide and trace water form the newly-generated inferior phosphide key of acid attack.

The 5th step----oxidation (Oxidation), the Nucleotide that newly adds after the ligation is gone up the oligonucleotide chain that links to each other by inferior phosphide key (phosphorus is trivalent) with CPG and is connected, therefore this inferior phosphide key instability easily by acid, basic hydrolysis, needs three valent phosphors herein to be oxidized to the phosphorus of pentavalent.Oxygenant commonly used is the tetrahydrofuran solution of iodine.This step speed of response is also very fast.Should note last circulation time, oxidation step can not omit.Also available in addition other oxygenants are finished oxidation, thereby obtain the various oligonucleotide that meet the experiment needs.

Circulate above-mentioned one to five step, can finish when oligonucleotide chain promptly may extend to desired length.

2. synthetic aftertreatment:

(1.) cutting: will synthesize good oligonucleotide chain chemical chop from the upholder and get off.Fresh strong aqua commonly used comes the last ester bond that connects between compound and initial nucleosides of cracking CPG.The oligonucleotide that ruptures has 3 ' hydroxyl freely.

(2.) deprotection base: must before this step, choose follow-up purification process.Generally handle the long period to take off nitrile ethyl, benzoyl, sec.-propyl, take off 5 '-DMT with trifluoroacetic acid with fresh strong aqua.

(3.) purifying: according to the composition of institute's synthetic oligonucleotide with should be used for the method for selected purifying.Purification process commonly used has: C18 post, OPC post, PAGE and HPLC.

(4.) quantitative.Come quantitatively in the uv-absorbing at 260nm place according to oligonucleotide.

(5.) store.Usually once the amount of the synthetic oligonucleotide that provides can be more much bigger than needed amount, so can relate to the storage problem of oligonucleotide.In general, this does not become problem, because oligonucleotide is stable fine.It should be noted that violent physical environments such as avoiding ultraviolet ray, avoid multigelation, can stablize for-20 ℃ and preserve more than 1 year.

Experimental example 3

Present embodiment is that the activity of the ODN inhibition CpG-S ODN of 7 no obvious irritation is identified.

Separate and cultivator peripheral blood mononuclear cell (hPBMC), adjusting the hPBMC concentration of cell suspension is 1 * 10 ⁶/ ml adds in 24 orifice plates, every hole 1ml.Put 37 ℃ of CO ₂Incubator adds 10 μ g/ml ODN respectively to be screened after 30 minutes after cultivating 2h, adds the CpG-S ODN (TGG CGC GGGGCG) of 5 μ g/ml again, puts 37 ℃ of CO ₂Incubator is got cells and supernatant after cultivating 4h, adopt the double-antibody sandwich elisa method to measure the concentration of TNF-α in the cells and supernatant, clear and definite above-mentioned 7 ODN suppress CpG-S ODN and induce hPBMC to discharge the ability of TNF-α, this time experimental result shows that above-mentioned 7 ODN all have inhibition CpG-S ODN to induce hPBMC to discharge the ability that TNF-α discharges, so we formally are referred to as CpG-N ODN (seeing Table 5).

Table 5.CpG-N ODN induces the human PBMC to discharge influence (n=6, the x ± s) of TNF-α to CpG-S ODN

Sequence number	TNF-α(pg/ml)	Inhibiting rate (%)
			Control group CpG-S ODN ODN1 ODN6 ODN15 ODN18 ODN19 ODN20 ODN21	108±7 2776±136 2133±242 ★2434±124 ★1012±342 ★957±272 ★1121±380 ★1252±144 ★1557±251 ★	- - 23.2 12.3 63.5 65.5 59.6 54.9 43.9

^★: compare p＜0.01 with CpG-S ODN group

Experimental example 4

Present embodiment is to study CpG-N ODN attacks mouse to CpG-S ODN provide protection.

Cleaning level 90 of the kind small white mouses in Kunming (Third Military Medical University's Experimental Animal Center provides), body weight 19.9 ± O.5g/ only, male and female are regardless of, and are divided into control group, CpG-S ODN, ODN1, ODN6, ODN15, ODN18, ODN19, ODN20, ODN21 group at random.Control group gives water for injection, and volume injected is 200 μ l/20g body weight.CpG-S ODN organizes, and gives the CpG-S ODN of 80 μ g/20g body weight; All the other each groups give 80 μ g/20g CpG-S ODN immediately after giving corresponding C pG-NODN 400 μ g/20g body weight; Administering mode is a tail vein injection, observes mouse generalized case and mortality ratio (table 6) in 7 days.It is all dead that the result shows that CpG-S ODN can cause in the mouse 3 days, and 7 CpG-N ODN can reduce CpG-S ODN and attack mortality of mice.

7 CpG-N ODN of table 6. attack the provide protection of mouse to CpG-S ODN

Group	Mouse diing time (my god) and quantity (only)							Mortality ratio (%)
									D1	D2	D3	D4	D5	D6	D7
	Control group CpG-S ODN ODN1 ODN6 ODN15 ODN18 ODN19 ODN20 ODN21	0 8 4 5 3 3 2 2 5	0 9 5 6 4 4 3 3 6	0 10 5 6 5 6 4 4 6	0 10 6 6 5 6 5 4 6	0 10 6 6 5 6 5 4 6	0 10 6 6 5 6 5 4 6									0 10 6 6 5 6 5 4 6	0(0/10) 100(10/10) 60(6/10) ★60(6/10) ★50(5/10) ★60(6/10) ★50(5/10) ★40(4/10) ★60(6/10) ★

★: compare p＜0.05 with CpG-S ODN group

SEQUENCE?LISTING

＜110〉Military Medical Univ No.3, P.L.A

＜120〉gene order and the application thereof of the CpG-N ODN of high immunological activity CpG-S ODN and antagonism CpG-S ODN effect

<130>2672-107

<140>200410034787.X

<141>2004-05-17

<160>21

<170>PatentIn?version?3.1

<210>1

<211>12

<212>DNA

＜213〉the unknown

<400>1

ggcggcggcg?cg 12

<210>2

<211>12

<212>DNA

＜213〉the unknown

<400>2

cgcggcgccg?gg 12

<210>3

<211>12

<212>DNA

＜213〉the unknown

<400>3

ggcggcggcg?ga 12

<210>4

<211>12

<212>DNA

＜213〉the unknown

<400>4

ccgcgcgcgg?gc 12

<210>5

<211>12

<212>DNA

＜213〉the unknown

<400>5

tggcgcgggg?cg 12

<210>6

<211>12

<212>DNA

＜213〉the unknown

<400>6

gccggcgccc?gg 12

<210>7

<211>12

<212>DNA

＜213〉the unknown

<400>7

gcgcgcgcgg?ta 12

<210>8

<211>12

<212>DNA

＜213〉the unknown

<400>8

ccaggcggcg?gg 12

<210>9

<211>12

<212>DNA

＜213〉the unknown

<400>9

ggccgcgggg?gt 12

<210>10

<211>12

<212>DNA

＜213〉the unknown

<400>10

cagcccgggg?ga 12

<210>11

<211>12

<212>DNA

＜213〉the unknown

<400>11

gctcccgggc?tt 12

<210>12

<211>12

<212>DNA

＜213〉the unknown

<400>12

gcgctcgggc?tt 12

<210>13

<211>12

<212>DNA

＜213〉the unknown

<400>13

ccgcccgcgc?ct 12

<210>14

<211>12

<212>DNA

＜213〉the unknown

<400>14

gcacccgcgc?gc 12

<210>15

<211>12

<212>DNA

＜213〉the unknown

<400>15

ggccgcgggg?ac 12

<210>16

<211>12

<212>DNA

＜213〉the unknown

<400>16

gccgccgccg?cc 12

<210>17

<211>12

<212>DNA

＜213〉the unknown

<400>17

cgcctcgggg?ag 12

<210>18

<211>12

<212>DNA

＜213〉the unknown

<400>18

tgccgcggca?ga 12

<210>19

<211>12

<212>DNA

＜213〉the unknown

<400>19

ctccacgtgc?cg 12

<210>20

<211>12

<212>DNA

＜213〉the unknown

<400>20

gccgccgccg?tc 12

<210>21

<211>12

<212>DNA

＜213〉the unknown

<400>21

gctgccgccg?cc 12

Claims (6)

1. the immunostimulatory oligonucleotide of a high immunological activity is characterized in that, has following gene order:

GGC?GGC?GGC?GCG；

CGC?GGC?GCC?GGG；

GGC?GGC?GGC?GGA；

CCG?CGC?GCG?GGC；

TGG?CGC?GGG?GCG；

GCC?GGC?GCC?CGG；

GCG?CGC?GCG?GTA；

CCA?GGC?GGC?GGG；

GGC?CGC?GGG?GGT；

CAG?CCC?GGG?GGA；

GCT?CCC?GGG?CTT；

GCG?CTC?GGG?CTT；

CCG CCC GCG CCT; Or

Wherein a kind of of GCACCC GCG CGC.

2. the immunostimulatory oligonucleotide of a kind of high immunological activity according to claim 1 is characterized in that, has following gene order:

GGC?GGC?GGC?GCG；

CCG?CGC?GCG?GGC；

TGG?CGC?GGG?GCG；

GCC?GGC?GCC?CGG；

GGC?CGC?GGG?GGT；

CAG?CCC?GGG?GGA；

GCT?CCC?GGG?CTT；

GCG CTC GGG CTT; Or

Wherein a kind of of CCG CCC GCG CCT.

3. the oligonucleotide to the immunostimulatory oligonucleotide antagonism is characterized in that, has following gene order:

GGC?CGC?GGG?GAC；

GCC?GCC?GCC?GCC；

CGC?CTC?GGG?GAG；

TGC?CGC?GGC?AGA；

CTC?CAC?GTG?CCG；

GCC GCC GCC GTC; Or

Wherein a kind of of GCT GCC GCC GCC.

4. the oligonucleotide to the immunostimulatory oligonucleotide antagonism according to claim 3 is characterized in that, has following gene order:

CGC?CTC?GGG?GAG；

TGC?CGC?GGC?AGA；

CTC?CAC?GTG?CCG；

GCC GCC GCC GTC; Or

Wherein a kind of of GCTGCC GCC GCC.

5. the immunostimulatory oligonucleotide of claim 1 or 2 described high immunological activities is in the application that is used for preparing treatment infectious diseases, anaphylactic disease, immunodeficiency diseases and tumour medicine.

6. claim 3 or 4 described oligonucleotide to the immunostimulatory oligonucleotide antagonism are being used for preparing the application for the treatment of SIRS and medication for treating pyemia.

Images