CN101979566B - Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof - Google Patents
Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof Download PDFInfo
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Abstract
The invention relates to sulpho-oligodeoxynucleotide with immunostimulation activities. The sequence of the sulpho-oligodeoxynucleotide has two or more than two copied 5'-NTCGTT-3' elements having 15 to 35 nucleotides respectively, wherein CpG is non-methylated and N does not represent A or G. In vitro, the sulpho-oligodeoxynucleotide has high immunostimulation activities for the immunocytes of human and a mouse, and the immunostimulation activities of the sulpho-oligodeoxynucleotide in a human body can be evaluated according to the result of the immunostimulation activities in mouse. Used as a vaccine adjuvant, the sulpho-oligodeoxynucleotide can obviously strengthen the immune response of the mouse to a genetic engineering hepatitis B vaccine and make the immune response deflected to a TH1 direction, and thus, it is proved that the sulpho-oligodeoxynucleotide can be used for preventing and treating allergy and infection. Meanwhile, the sulpho-oligodeoxynucleotide has high tumor growth restraining activities in the mouse body.
Description
The application be that January 23, application number in 2008 are 200810004736.0 the applying date, denomination of invention divides an application for the application of " sulpho-oligodeoxynucleotidewith and application thereof with immunostimulatory activity ".
Technical field
The present invention relates to biological field, specifically, relate to a kind of sulpho-oligodeoxynucleotidewith and application thereof with immunostimulatory activity.
Background technology
Last century, the eighties Japanese scholars found that the DNA component in the bacille Calmette-Guerin vaccine (BCG) has anti-tumor activity in the humans and animals body, and the scientist who studies afterwards the antisense nucleic acid treatment finds that again there is non-specific immunostimulatory activity in the sulpho-oligodeoxynucleotidewith of synthetic.Krieg AM etc. are at " Nature " 1995, disclosed the essence of these phenomenons in the 374th volume, the 546-549 page or leaf, find immunostimulatory activity composition in the nucleic acid be some contain non-ly methylate 5 '-the Hexanucleotide sequence of CpG-3 ' dinucleotides, (Pu represents purine A or G to be characterized as 5 '-PuPuCpGPyPy-3 '; Py represents pyrimidine C or T), such as 5 '-GACGTT-3 ' etc.Wherein, core Nucleotide C and G are active essential by it, and each two Nucleotide of two ends also are closely related with its activity.Above-mentioned Hexanucleotide characteristic sequence is called CpG primitive (CpG motif), and the oligodeoxynucleotide that contains the CpG primitive is referred to as CpG oligodeoxynucleotide (CpG-ODN).The CpG-ODN that contains above-mentioned feature has good immunostimulatory activity to mouse, and results of animal has shown that CpG-ODN has a good application prospect in fields such as vaccine adjuvant, tumor prevention and treatment, infection mitigation and treatment and irritated prevention and treatments.But further research finds that its immunostimulatory activity has species specificity, and the CpG primitive and the CpG-ODN that therefore illustrate activation people immunocyte are the prerequisites that CpG-ODN is applied to the human clinical.Krieg AM etc. " are reporting that first can activate CpG the primitive 5 '-GTCGTT-3 ' of people's immunocyte in The Journal of Immunology 2000, the 164 volumes, the 1617-1624 page or leaf.Derivative CpG-ODN (such as CpG-ODN 2006) has good immunostimulatory activity external to people's immunocyte thus, and present a plurality of human clinical's research has also confirmed the immune effect of CpG-ODN 2006 in fields such as vaccine adjuvant, oncotherapy and treatment of infection.According to results of study such as Krieg AM, above-mentioned CpG-ODN is that the people is special, mouse there is not activity, therefore can only estimate these sequences immunostimulatory activity in vivo as animal model with primates such as monkeys, this has increased the difficulty of experiment, is difficult to obtain such as cost increase, animal-origin etc.Therefore, the wide spectrum CpG-ODN that finds a kind of non-species specificity extremely is necessary.
For the research of relevant CpG-ODN in document " Chinese microbiology and IMMUNOLOGY KEY WORDS INDEX 2001,21 (5), 471-475 page or leaf, " animal medicine progress " 2004,25 (4), 25-28 page or leaf, " Gene Therapy " 1996, the 6 volumes are described in the 893-903 page or leaf to some extent.In addition, number of C pG-ODN and relevant application thereof are also disclosed among patent documentation CN 1271733A, CN 1526718A, US 2005/0267057A1 or the WO 99/56755, but in above-mentioned document, all unexposed relevant Nucleotide with 5 ' of two or more copies-NTCGTT-3 ' primitive (N does not represent A or G), also unexposed any technology enlightenments that people and mouse is had the cross immunity stimulating activity about the sulpho-oligodeoxynucleotidewith that comprises this primitive.
Summary of the invention
The object of the present invention is to provide sulpho-oligodeoxynucleotidewith a kind of non-species specificity, that have immunostimulatory activity.More particularly, the invention provides a kind of sulpho-oligodeoxynucleotidewith that people and mouse is had the cross immunity stimulating activity.
The present invention also aims to provide this sulpho-oligodeoxynucleotidewith with immunostimulatory activity in preparation vaccine adjuvant and the application in the medicine of preparation prevention or treatment tumour, infection and allergy.
The inventor studies on the basis of existing technology, finds: the CpG primitive of (1) activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T); (2) the CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).On this Research foundation, find out that the CpG-ODN that contains 5 '-NTCGTT-3 ' primitive (comprising 5 '-ATCGTT-3 ', 5 '-GTCGTT-3 ', 5 '-CTCGTT-3 ' and 5 '-TTCGTT-3 ') has the cross immunity stimulating activity to people and mouse.The contriver further studies show that, having 5 ' of two or more copies-NTCGTT-3 ' primitive (N does not represent A or G) in the nucleotide sequence is sulpho-oligodeoxynucleotidewith of the present invention has the cross immunity stimulating activity to people and mouse key.
Given this, the invention provides a kind of sulpho-oligodeoxynucleotidewith with immunostimulatory activity, have 5 ' of two or more copies-NTCGTT-3 ' primitive in its sequence, described N does not represent A or G, is preferably T or C.The length of described sulpho-oligodeoxynucleotidewith is 15～35 Nucleotide, is preferably 20～30 Nucleotide.Described CpG right and wrong are methylated.
When N was T, the sequence preference of described sulpho-oligodeoxynucleotidewith was:
5’-TCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTCGTTCGTTCGTTCGTTCGTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TCGTTTCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTTTTCGTTTTTCGTT-3’、
5’-TTCGTTATTCGTTATTCGTT-3’、
5’-TTCGTTGTTCGTTGTTCGTT-3’、
5’-TTCGTTCTTCGTTCTTCGTT-3’、
5’-TCTTCGTTTTTCGTTTTTCGTT-3’、
5’-TCTTCGTTATTCGTTATTCGTT-3’、
5’-TCTTCGTTGTTCGTTGTTCGTT-3’、
5’-TCTTCGTTCTTCGTTCTTCGTT-3’、
5’-TTCGTTTTCGTTTTCGTTTTCGTT-3’、
5’-TCTTCGTTTTCGTTTTCGTT-3’、
5’-TTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTTCGTTTCGTTTCGTTTCGTT-3’、
5’-TTCGTTCGTTCGTTCGTT-3’、
5’-TCTTCGTTCGTTCGTTCGTT-3’、
5 '-TTCGTTCGTTCGTTCGTTCGTT-3 ' or
5’-TTCGTTCGTTCGTTCGTTCGTTCGTT-3’。
When described N was C, the sequence preference of described sulpho-oligodeoxynucleotidewith was:
5 '-TCGTCTCGTTTCGTCTCGTT-3 ' or
5’-TCGTCTCGTTTCGTCTCGTTTCGTCTCGTT-3’。
The preparation method of sulpho-oligodeoxynucleotidewith of the present invention is well-known to those skilled in the art, for example can adopt the chemosynthesis of solid phase phosphoramidite triester method, this method has efficient and the advantage such as quick coupling, has been widely used in DNA the field of chemical synthesis.Entrust gene Synesis Company of specialty to process for the oligodeoxynucleotide synthetic at present, for example the domestic Shanghai living worker biotech company that entrusts is synthetic more.
The solid phase phosphoramidite triester method is by 3 ' end beginning, and concrete reactions steps is as follows:
1. Deprotection: slough the blocking group DMT (dimethoxytrityl) that is connected to the Nucleotide on the CPG (Controlled Pore Glass) with trichoroacetic acid(TCA), obtain 5 ' free hydroxyl, use for next step condensation reaction;
2. activation: with nucleotide monomer and the tetrazole activator mix of phosphoramidite protection and enter synthetic post, (its 3 ' end is activated to form phosphoramidite tetrazolium active intermediate, but 5 ' end is protected by DMT still), the Nucleotide generation condensation reaction of Deprotection on this intermediate and the CPG;
3. connect: when phosphoramidite tetrazolium active intermediate runs on the CPG the Nucleotide of Deprotection, will with its 5 ' hydroxyl generation affinity reaction, condensation is also sloughed tetrazolium, this moment, oligonucleotide chain prolonged forward a base;
4. oxidation: nucleotide monomer is to be connected with oligonucleotide on being connected in CPG by inferior phosphide key during condensation reaction, and inferior phosphide key is unstable, easily by acid or basic hydrolysis, use this moment sulfo-reagent phosphoramidite to be oxidized to the phosphotriester of the two keys of sulphur phosphorus, thereby obtain stable oligonucleotide;
5. sealing: for the 5 ' hydroxyl that has neither part nor lot in reaction that prevents from being connected on the CPG is extended, often seal this terminal hydroxy group by acetylize after the condensation reaction in circulating reaction subsequently.
Through behind the above five steps, a deoxynucleotide just is connected on the Nucleotide of CPG.Repeat above Deprotection, activation, connection, oxidation, closed process and can obtain a dna fragmentation crude product.At last to it cuts, Deprotection, purifying (commonly used have the methods such as PAGE method, HPLC method, C18 method and OPC method), the synthetic aftertreatment such as quantitative can obtain meeting requirement of the present invention sulpho-oligodeoxynucleotidewith.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the preparation vaccine adjuvant.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the medicine of preparation prevention or treatment tumour.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the medicine of preparation prevention or treatment infection.
The present invention also provides the application of sulpho-oligodeoxynucleotidewith in the medicine of preparation prevention or treatment allergy.
Described sulpho-oligodeoxynucleotidewith all has good immunostimulatory activity external to people and mouse immune cell, can be from the immunostimulatory activity result the mouse is estimated its immunostimulatory activity in human body.As vaccine adjuvant, it can significantly strengthen mouse to the immunne response of recombinant hepatitis b vaccine, and can make immunne response deflection TH
1Direction.Because sulpho-oligodeoxynucleotidewith of the present invention can induce out very strong TH
1Para-immunity is replied, and shows that it also can be used for prevention and treatment irritated and that infect.Simultaneously, sulpho-oligodeoxynucleotidewith of the present invention also has the activity of good inhibition tumor growth in Mice Body.
Description of drawings
The figure of people's immunostimulatory activity of the oligodeoxynucleotide of Fig. 1 has 5 '-NTCGTPy-3 ' for illustrating (wherein N represents A, G, C or T, and Py represents pyrimidine C or T) primitive.
The figure of the mouse immune stimulating activity of the oligodeoxynucleotide of Fig. 2 has 5 '-NA/TCGTT-3 ' for illustrating (wherein N represents A, G, C or T) primitive.
Fig. 3 a is the figure that people's immunostimulatory activity of oligodeoxynucleotide of the present invention (T1～T6, C1 and C2) and control sequence 1826 is shown.
Fig. 3 b is for illustrating the oligodeoxynucleotide of the present invention (figure of people's immunostimulatory activity of T7～T14).
Fig. 3 c is for illustrating the oligodeoxynucleotide of the present invention (figure of people's immunostimulatory activity of T15～T22).
Fig. 4 a is the figure that the mouse immune stimulating activity of oligodeoxynucleotide of the present invention (T1～T6, C1 and C2) and control sequence 1826 and T7C is shown.
Fig. 4 b is for illustrating the oligodeoxynucleotide of the present invention (figure of the mouse immune stimulating activity of T7～T14).
Fig. 4 c is for illustrating the oligodeoxynucleotide of the present invention (figure of the mouse immune stimulating activity of T15～T22).
Fig. 5 illustrates oligodeoxynucleotide of the present invention to strengthen mouse to the figure of the immunne response of genetically engineered hepatitis B surface antigen as vaccine adjuvant.
Fig. 6 illustrates oligodeoxynucleotide of the present invention and other vaccine adjuvant coupling to strengthen mouse to the figure of the immunne response of genetically engineered hepatitis B surface antigen.
Fig. 7 induces TH for oligodeoxynucleotide of the present invention is shown in Mice Body
1The figure that para-immunity is replied.
Fig. 8 illustrates oligodeoxynucleotide of the present invention suppresses tumor growth in Mice Body figure.
Embodiment
The invention will be further described below in conjunction with the description of accompanying drawing by embodiment, but this is not to be limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within the scope of the present invention.
In order to illustrate the CpG primitive of activation people immunocyte, the inventor has carried out mutation research to four Nucleotide of all the other except core Nucleotide CpG in the CpG primitive.For this reason, the contriver has designed one group of CpG-ODN, and is as shown in table 1, and every sequence length is 12 Nucleotide, all contains the CpG primitive of two identical copies.Stimulate the human peripheral blood single nucleus cell (PBMC) of vitro culture with these CpG-ODN, by measuring the IgM content in the culture supernatant, can estimate them to the activity of people's immunocyte.
Employed CpG-ODN is designed by the inventor in the present embodiment, entrust Shanghai to give birth to worker biotech company and carry out synthetic, and carry out full chain thio-modification, polyacrylamide gel electrophoresis (PAGE) purifying, be dissolved in physiological saline, save backup in-20 ℃ of refrigerators.
Table 1
| Sequence | Numbering | Sequence | |
| 1 | 1444 | 5’-ATCGTT ATCGTT-3’ | |
| 2 | 2444 | 5’-GTCGTT GTCGTT-3’ | |
| 3 | 3444 | 5’-CTCGTT CTCGTT-3’ | |
| 4 | 4444 | 5’-TTCGTT TTCGTT-3’ | |
| 5 | 2144 | 5’-GACGTT GACGTT-3’ | |
| 6 | 2244 | 5’-GGCGTT GGCGTT-3’ | |
| 7 | 2344 | 5’-GCCGTT GCCGTT-3’ | |
| 8 | 2444 | 5’-GTCGTT GTCGTT-3’ | |
| 9 | 2414 | 5’-GTCGAT GTCGAT-3’ | |
| 10 | 2424 | 5’-GTCGGT GTCGGT-3’ | |
| 11 | 2434 | 5’-GTCGCT GTCGCT-3’ | |
| 12 | 2444 | 5’-GTCGTT GTCGTT-3’ | |
| 13 | 2441 | 5’-GTCGTA GTCGTA-3’ | |
| 14 | 2442 | 5’-GTCGTG GTCGTG-3’ | |
| 15 | 2443 | 5’-GTCGTC GTCGTC-3’ | |
| 16 | 2444 | 5’-GTCGTT GTCGTT-3’ |
Get Healthy People venous blood under the aseptic condition, employment lymphocyte separation medium behind the anticoagulant heparin (available from the same positive biotech company in the north) separates PBMC; With RPMI 1640 substratum (available from U.S. Gibco company) cell concn is adjusted to 1 * 10 behind the counting
6/ ml is seeded in the 96 porocyte plates, every hole 200 μ l; Adding CpG-ODN is 2 μ M to final concentration, 5%CO
2The lower 37 ℃ of cultivations of condition 8 days.With the coated 96 hole enzyme plates of goat-anti people IgM (available from U.S. SIGMA company), every hole 200ng, 4 ℃ are spent the night; Add above-mentioned PBMC culture supernatant after washing plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash the goat-anti people IgM (available from U.S. SIGMA company) that plate adds the horseradish peroxidase-labeled of dilution in 1: 10000 for 3 times afterwards, every hole 100 μ l, 37 ℃ act on 1 hour; Use O-Phenylene Diamine (OPD) colour developing after washing plate 3 times, 2M sulfuric acid termination reaction is with the spectrophotometric determination 490nm absorbance OD490 of place.Measured OD490 value is larger, shows that this CpG-ODN is stronger to the activity of people's immunocyte.
The results detailed in Fig. 1, wherein " contrast " be not for adding the cell contrast of CpG-ODN.Sequence number is four sequences of 1～4, and only in first Nucleotide difference of CpG primitive, the result shows that they are suitable to the activity of people's immunocyte, shows that first Nucleotide of the CpG primitive of activation people immunocyte is N (wherein N represents A, G, C or T); Sequence number is four sequences of 5～8, and only different at the second Nucleotide of CpG primitive, the result shows that this position is necessary for T, otherwise very weak to the activity of people's immunocyte; Sequence number is four sequences of 9～12, and only in the 5th Nucleotide difference of CpG primitive, the result shows that this position also is necessary for T, otherwise very weak to the activity of people's immunocyte; Sequence number is four sequences of 13～16, and only in the 6th Nucleotide difference of CpG primitive, the result shows that this position has preferably immunostimulatory activity when being C or T.In sum, the CpG primitive of activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T).
In order to illustrate the CpG primitive of activation mouse immune cell, the inventor has carried out mutation research to four Nucleotide of all the other except core Nucleotide CpG in the CpG primitive.The CpG-ODN sequence of using in the present embodiment sees Table 1.Stimulate mouse spleen mononuclearcell (SMMC) with these CpG-ODN, by measuring the IgM content in the culture supernatant, can estimate them to the activity of mouse immune cell.Employed is the Balb/c mouse, female, and 6～8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
Get the Balb/c mouse spleen under the aseptic condition, prepare the SMMC suspension with RPMI 1640 substratum; With RPMI 1640 substratum cell concn is adjusted to 1 * 10 behind the counting
6/ ml is seeded in the 96 porocyte plates, every hole 200 μ l; Adding CpG-ODN is 2 μ M to final concentration, 5%CO
2The lower 37 ℃ of cultivations of condition 3 days.With the coated 96 hole enzyme plates of goat-anti Mouse IgM (available from U.S. SIGMA company), every hole 200ng, 4 ℃ are spent the night; Add above-mentioned SMMC culture supernatant after washing plate 3 times, every hole 100 μ l, 37 ℃ of effects 1 hour; Wash the goat-anti Mouse IgM (available from U.S. SIGMA company) that adds the horseradish peroxidase-labeled of dilution in 1: 10000 behind the plate 3 times, 1,37 ℃ of effect of every hole 100 μ 1 hour; Wash behind the plate 3 times with the OPD colour developing, 2M sulfuric acid termination reaction is with the spectrophotometric determination 490nm absorbance OD490 of place.Measured OD490 value is larger, shows that this CpG-ODN is stronger to the activity of mouse immune cell.
The results detailed in Fig. 2, wherein " contrast " be not for adding the cell contrast of CpG-ODN.Sequence number is four sequences of 1～4, only in first Nucleotide difference of CpG primitive, the result shows that they are suitable to the activity of mouse immune cell, shows that first Nucleotide of the CpG primitive of activation mouse immune cell is N (wherein N represents A, G, C or T); Sequence number is four sequences of 5～8, and only different at the second Nucleotide of CpG primitive, the result shows that this position is necessary for A or T, otherwise very weak to the activity of mouse immune cell; Sequence number is four sequences of 9～12, and only in the 5th Nucleotide difference of CpG primitive, the result shows that this position is necessary for T, otherwise very weak to the activity of mouse immune cell; Sequence number is four sequences of 13～16, and only in the 6th Nucleotide difference of CpG primitive, the result shows that this position also is necessary for T, otherwise very weak to the activity of mouse immune cell.In sum, the CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).
By the result of study of embodiment 1 and 2 as can be known, the CpG primitive of activation people immunocyte is 5 '-NTCGTPy-3 ' (wherein N represents A, G, C or T, and Py represents pyrimidine C or T); The CpG primitive of activation mouse immune cell is 5 '-NA/TCGTT-3 ' (wherein N represents A, G, C or T).Comprehensive above-mentioned result of study can find out that the CpG primitive that people and mouse are had a cross immunity stimulating activity is 5 '-NTCGTT-3 ' (wherein N represents A, G, C or T).
Embodiment 3. oligodeoxynucleotide of the present invention are to the immunostimulatory activity of people's immunocyte
According to what find in embodiment 1 and 2 people and mouse had the CpG primitive of cross immunity stimulating activity, the contriver designs and has synthesized one group of CpG-ODN, as shown in table 2, length is 15～35 Nucleotide, contains respectively two or (be numbered T1～T22) and 5 '-CTCGTT-3 ' primitive (being numbered C1 and C2) more than 5 '-TTCGTT-3 ' primitive of two copies.
CpG-ODN in the table 2 draws respectively from document " Chinese microbiology and IMMUNOLOGY KEY WORDS INDEX 2001 except control sequence T7C and 1826,21 (5), 471-475 page or leaf, " The Journal of Immunology " 1998, the 160th volume, the 870-876 page or leaf, all the other design by the inventor.The sequence of used control sequence and contriver's designed, designed all entrusts Shanghai living worker biotech company to synthesize in the present embodiment, full chain thio-modification, and the PAGE purifying is dissolved in physiological saline, saves backup in-20 ℃ of refrigerators.
Table 2
| Numbering | Sequence |
| T1 | 5’-TCGTTCGTTCGTTCGTTCGTT-3’ |
| T2 | 5’-TCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
| T3 | 5’-TCGTTCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
| T4 | 5’-TCGTTTCGTTTCGTTTCGTT-3’ |
| T5 | 5’-TCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
| T6 | 5’-TCGTTTCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
| T7 | 5’-TTCGTTTTTCGTTTTTCGTT-3’ |
| T8 | 5’-TTCGTTATTCGTTATTCGTT-3’ |
| T9 | 5’-TTCGTTGTTCGTTGTTCGTT-3’ |
| T10 | 5’-TTCGTTCTTCGTTCTTCGTT-3’ |
| T11 | 5’-TCTTCGTTTTTCGTTTTTCGTT-3’ |
| T12 | 5’-TCTTCGTTATTCGTTATTCGTT-3’ |
| T13 | 5’-TCTTCGTTGTTCGTTGTTCGTT-3’ |
| T14 | 5’-TCTTCGTTCTTCGTTCTTCGTT-3’ |
| T15 | 5’-TTCGTTTTCGTTTTCGTTTTCGTT-3’ |
| T16 | 5’-TCTTCGTTTTCGTTTTCGTT-3’ |
| T17 | 5’-TTCGTTTCGTTTCGTTTCGTT-3’ |
| T18 | 5’-TTCGTTTCGTTTCGTTTCGTTTCGTT-3’ |
| T19 | 5’-TTCGTTCGTTCGTTCGTT-3’ |
| T20 | 5’-TCTTCGTTCGTTCGTTCGTT-3’ |
| T21 | 5’-TTCGTTCGTTCGTTCGTTCGTT-3’ |
| T22 | 5’-TTCGTTCGTTCGTTCGTTCGTTCGTT-3’ |
| C1 | 5’-TCGTCTCGTTTCGTCTCGTT-3’ |
| C2 | 5’-TCGTCTCGTTTCGTCTCGTTTCGTCTCGTT-3’ |
| T7C | 5’-TCGTCGTCGTCGTCGTCGTCG-3’ |
| 1826 | 5’-TCCATGACGTTCCTGACGTT-3’ |
Measure oligodeoxynucleotide of the present invention to the immunostimulatory activity of people's immunocyte according to embodiment 1 described method, difference is that adding oligodeoxynucleotide of the present invention to final concentration in 96 porocyte plates is 4nM, 8nM, 16nM and 32nM.The results are shown in Table 3, Fig. 3 a, 3b and 3c.
Table 3
By table 3, Fig. 3 a, 3b and 3c as seen, oligodeoxynucleotide of the present invention has the immunostimulatory activity of height to people's immunocyte, very strong activity is just arranged in 4～32nM concentration range.Control sequence 1826 contains two copies 5 '-GACGTT-3 ' primitive, is mouse specific C pG-ODN, therefore to the activity of people's immunocyte a little less than.
According to embodiment 2 described methods, measure oligodeoxynucleotide of the present invention to the immunostimulatory activity of mouse immune cell, difference is that adding oligodeoxynucleotide of the present invention to final concentration in 96 porocyte plates is 12.5nM, 25nM, 50nM, 100nM and 200nM.The results are shown in Table 4, Fig. 4 a, 4b and 4c.
Table 4
By table 4, Fig. 4 a, 4b and 4c as seen, oligodeoxynucleotide of the present invention has the immunostimulatory activity of height to the mouse immune cell, very strong activity is just arranged in 12.5～200nM concentration range.Control sequence 1826 contains two copies 5 '-GACGTT-3 ' primitive, is mouse specific C pG-ODN, therefore the mouse immune cell is had the strongest activity.Control sequence T7C contains multiple copied 5 '-GTCGTC-3 ' primitive, is people's specific C pG-ODN, and is therefore very weak to the activity of mouse immune cell.
Can be found out by the experimental result in embodiment 3 and 4, can design the oligodeoxynucleotide that people and mouse is had height cross immunity stimulating activity according to CpG the primitive 5 '-NTCGTT-3 ' (wherein N does not represent A or G) that people and mouse is had the cross immunity stimulating activity, also prompting can be estimated with mouse potential human clinical's using value of this class oligodeoxynucleotide as animal model simultaneously.
Embodiment 5. oligodeoxynucleotide of the present invention strengthen mouse to the immunne response of genetically engineered hepatitis B surface antigen as vaccine adjuvant
Use CpG-ODN T1～T6 and C1～C2 in the present embodiment.Genetically engineered hepatitis B surface antigen (HBsAg) is provided with epi chamber by Virology Inst., Chinese Academy of Preventive Medical Science's virus heredity, expressing cho cell, and purity is greater than 95%.The Balb/c mouse, female, 6～8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
HBsAg is adsorbed in the Al (OH) of 1mg/ml
3, final protein concentration is 10 μ g/ml.Through left hind gastrocnemius muscle immunity Balb/c mouse, cumulative volume is 100 μ l, every group of 6 mouse.Every injected in mice of experimental group 1 μ g is through Al (OH)
3HBsAg and the 10 μ g oligodeoxynucleotide of the present invention of absorption, every injected in mice of control group 1 μ g is through Al (OH)
3The HBsAg of absorption.The rear 4 weeks blood sampling of immunity is also isolated serum, according to ordinary method this serum is carried out 2 times of serial dilutions, for detection of the total antibody of antigen-specific IgG.
With the coated 96 hole enzyme plates of HBsAg, every hole 200ng, 4 ℃ are spent the night; Washing plate sealed 1 hour with 37 ℃ of 1% bovine serum albumins for 3 times afterwards; Wash the serum to be checked that plate adds above-mentioned 2 times of serial dilutions for 3 times afterwards, 37 ℃ act on 1 hour; Wash the goat anti-mouse igg (available from U.S. SIGMA company) that plate adds the horseradish peroxidase-labeled of dilution in 1: 10000 for 3 times afterwards, every hole 100 μ l, 37 ℃ act on 1 hour; Wash behind the plate 3 times with the OPD colour developing, 2M sulfuric acid termination reaction is with the also definite terminal point titre (threshold value is made as 0.10) of the absorbance OD490 of spectrophotometric determination 490nm place.
" contrast " expression Al (OH) among Fig. 5
3As the HBsAg specific IgG antibodies titre that induces behind the adjuvant immunity.When immunity adds CpG-ODN of the present invention and can significantly strengthen immunne response to HBsAg, and specific antibody titre (logarithmic value) can strengthen more than 10 times, shows that they have a fabulous vaccine adjuvant active.Because these CpG-ODN have the cross immunity stimulating activity to people and mouse, point out them also to can be used as Human vaccine adjuvant.
Embodiment 6. oligodeoxynucleotide of the present invention and other vaccine adjuvant coupling strengthen mouse to the immunne response of genetically engineered hepatitis B surface antigen
Use CpG-ODN T1 in the present embodiment.Recombinant HBsAg is provided with epi chamber by Virology Inst., Chinese Academy of Preventive Medical Science's virus heredity, expressing cho cell, and purity is greater than 95%.The Balb/c mouse, female, 6～8 weeks are available from Chinese Academy of Medical Sciences's Experimental Animal Center.
With HBsAg directly with normal saline dilution or be adsorbed in the Al (OH) of 1mg/ml
3On, final protein concentration is 10 μ g/ml.Through left hind gastrocnemius muscle immunity Balb/c mouse, cumulative volume is 100 μ l, every group of 6 mouse.Every injected in mice 1 μ g of HBsAg group does not contain the HBsAg of any adjuvant, and every injected in mice 1 μ g is through Al (OH) for the HBsAg+Al group
3The HBsAg of absorption, the HBsAg of every injected in mice 1 μ g of HBsAg+CpG group and the CpG-ODN T1 of 10 μ g, every injected in mice 1 μ g is through Al (OH) for the HBsAg+Al+CpG group
3The HBsAg of absorption and the CpG-ODN T1 of 10 μ g.4 weeks blood sampling after the immunity, and according to the total antibody of embodiment 5 described method detectable antigens specific IgGs.
As seen from Figure 6, do not use the HBsAg group immune effect of adjuvant the poorest, the specific antibody titre only is 2.7 logarithmic value, with Al (OH)
3Or CpG-ODN T1 can significantly strengthen the immune effect of HBsAg as adjuvant, about specific antibody titre (logarithmic value) all can increase by 500.Al (OH)
3Have synergy with CpG-ODN T1, unite use and have the strongest adjuvanticity, can make the specific antibody titre increase more than 10 times.The above results shows, oligodeoxynucleotide of the present invention both can use separately as vaccine adjuvant, also can with other vaccine adjuvant such as Al (OH)
3The associating use.
Embodiment 7. oligodeoxynucleotide of the present invention strengthen TH in mouse
1Para-immunity is replied
According to embodiment 5 described methods, measure oligodeoxynucleotide of the present invention to mouse TH
1The impact that para-immunity is replied, employed enzyme labelled antibody was the goat anti-mouse igg 2a (available from U.S. SBA company) of horseradish peroxidase-labeled when difference was to detect.The result is presented among Fig. 7.
Antigen-specific immune response is divided into TH
1And TH
2Two types, TH wherein
1Class is replied corresponding with high-caliber antigen-specific IgG2a antibody titers.Al (OH)
3A kind of extremely strong TH
2The class vaccine adjuvant can suppress TH
1Para-immunity is replied, and induces extremely low-level specific IgG 2a antibody after showing as immunity.In the present embodiment, Al (OH)
3The specific IgG 2a antibody titers that induces as the HBsAg adjuvant only is 2.3 logarithmic value, shown in " contrast " among Fig. 7.Even add CpG-ODN immunne response deflection TH of the present invention during immunity
1Direction, showing as specific IgG 2a antibody titers increases by 2～3 logarithmic value, namely 100～1000 times.The above results shows, oligodeoxynucleotide of the present invention is the extremely strong TH of a class
1The para-immunity stimulant.Immunization Update studies show that, allergy is a kind of TH
2Para-immunity is replied relevant disease, is TH if type of immune response can be reversed
1Allergy can be prevented and treat to class.In addition, the prevention of tumour and infection and treatment all need to induce stronger TH
1Para-immunity is replied.Can induce very strong TH based on CpG-ODN of the present invention
1Therefore para-immunity is replied, and CpG-ODN of the present invention is except can be as vaccine adjuvant, also can be applicable to other fields such as the prevention of allergy, tumour and infection and treatment.
Embodiment 8. oligodeoxynucleotide of the present invention can suppress tumor growth in Mice Body
C-26 solid tumor (being provided by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences) is ground with copper mesh, is that 7.2 phosphate buffered saline buffer is adjusted to 1 * 10 with cell concn with pH
6/ ml, through left hind veutro subcutaneous vaccination Balb/c mouse, every mouse 100 μ l, every group of 8 mouse.From inoculating rear second day, per two days to tumor inoculation position injection CpG-ODN T1, every mouse 10 μ g, and volume is 100 μ l, control group mice is only injected 100 μ l physiological saline, altogether injects 6 times.The last injection was put to death mouse after 5 days, and the excision tumour is also weighed.The average knurl of calculating every treated animal is heavy, and calculates as follows tumour inhibiting rate: (the average knurl of the average knurl weight-treatment group of control group is heavy)/average knurl of control group heavy * 100%.The result is presented among Fig. 8.
As seen from Figure 8, the average knurl of control group mice heavily is 1.43 grams, and after 6 CpG-ODN T1 treatments of injection, the average knurl for the treatment of group mouse heavily is 0.59 gram, inhibitory rate to 58.7%.The above results shows, oligodeoxynucleotide of the present invention has good antitumous effect, can be used for clinically tumor immunotherapy.
Claims (6)
1. oligodeoxynucleotide with full chain thio-modification of immunostimulatory activity, has 5 ' of two or more copies-NTCGTT-3 ' primitive in its sequence, and its length is 15～35 Nucleotide, and wherein said CpG right and wrong are methylated, and described N is C.
2. the oligodeoxynucleotide of full chain thio-modification as claimed in claim 1, the length that it is characterized in that the oligodeoxynucleotide of described full chain thio-modification is 20～30 Nucleotide.
3. the oligodeoxynucleotide of full chain thio-modification as claimed in claim 1 or 2 is characterized in that the sequence of the oligodeoxynucleotide of described full chain thio-modification is:
5 ,-TCGTCTCGTTTCGTCTCGTT-3 ' or
5，-TCGTCTCGTTTCGTCTCGTTTCGTCTCGTT-3’。
4. a pharmaceutical composition is characterized in that being made by oligodeoxynucleotide and the pharmaceutically acceptable carrier of the described full chain thio-modification of claim 1～3 any one.
5. the application of the oligodeoxynucleotide of the described full chain thio-modification of claim 1～3 any one in the preparation vaccine adjuvant.
6. the application of the oligodeoxynucleotide of the described full chain thio-modification of claim 1～3 any one in the medicine for preparing prevention or treatment tumour, infection, allergy.
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| CN1271733A (en) * | 2000-04-04 | 2000-11-01 | 中国预防医学科学院病毒学研究所 | CpG oligonucleotide with specific immunostimulation activity to human immune cell |
| US6379661B1 (en) * | 1985-08-23 | 2002-04-30 | Amen Inc. | Pharmaceutical compositions comprising pluripotent granulocyte colony-stimulating factor |
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| US6379661B1 (en) * | 1985-08-23 | 2002-04-30 | Amen Inc. | Pharmaceutical compositions comprising pluripotent granulocyte colony-stimulating factor |
| CN1271733A (en) * | 2000-04-04 | 2000-11-01 | 中国预防医学科学院病毒学研究所 | CpG oligonucleotide with specific immunostimulation activity to human immune cell |
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Effective date of registration: 20151224 Address after: 100024 Beijing, Chaoyang District, the south of the room 4, No. three Patentee after: Xu Honglin Address before: 518000, Shenzhen, Guangdong province Baoan District Songgang Street Yan Chuan community factory building 2 (cattle horn road 5-1) Patentee before: Anqun Bioengineering Co., Ltd., Shenzhen |
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Effective date of registration: 20180420 Address after: 215000 room 810, building C, Cao Hu science and Technology Park, 1 West Kwun Tong, Xiangcheng Economic Development Zone, Suzhou, Jiangsu. Patentee after: Suzhou Bote dragon Immune Technology Co. Ltd. Address before: 100024 Beijing, Chaoyang District, the south of the room 4, No. three Patentee before: Xu Honglin |
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