CN1271733A - CpG oligonucleotide with specific immunostimulation activity to human immune cell - Google Patents
CpG oligonucleotide with specific immunostimulation activity to human immune cell Download PDFInfo
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Abstract
The present invention discloses a CpG oligonucleotides with specific immunostimulation activity to human immune cells, which can be used for preventing and curing human infective diseases, anaphylactic reaction, immunodeficiency and tumor. They are artificially synthesized non-methylated thiooligonucleotides with length of 10-40 bases and containing one or several copied human specific CpG characteristic sequence.
Description
Find that as far back as the eighties Japan scientist the DNA component of bacille Calmette-Guerin vaccine has anti-tumor activity in the humans and animals body
[1], the scientist of the early 1990s research antisense nucleic acid treatment finds that again there is non-specific immunostimulatory activity in artificial synthetic G 3139
[2]Nineteen ninety-five Krieg etc. has disclosed the essence of above-mentioned phenomenon, find that the immunostimulatory activity composition in the nucleic acid is some hexabasic basic characteristic sequences that contain 5 '-CG-3 ' dinucleotides that do not methylate, as 5 '-GACGTT-3 ', 5 '-GACGTC-3 ' and 5 '-GGCGTT-3 ' etc., core base C and G are by active essential, and the two ends base also is closely related with its activity
[3]Sato in 1996 etc. find that again the hexabasic basic characteristic sequence of 5 '-AACGTT-3 ' on the plasmid DNA can obviously strengthen dna vaccination at the intravital immune effect of mouse
[4]Because these oligonucleotide all contain 5 '-CG-3 ' dinucleotides, so be referred to as CpG oligonucleotide (CpG-ODN).
Functional study shows at external CpG-ODN has following activity to the mouse immune cell: (1) activation monokaryon, scavenger cell and dendritic cell, induce IFN-α β, and cytokines such as IL-12 and IL-18 promote B
7Expression with MHC II quasi-molecule; (2) cytokine activation NK cells such as collaborative IFN-α β and IL-12 strengthen its killing activity, induce IFN-γ; (3) polyclone activating B cell promotes the propagation and the differentiation of B cell, promotes the generation of immunoglobulin (Ig), promotes B
7Expression with MHC II quasi-molecule.
Therefore, CpG-ODN can be used as a kind of vaccine adjuvant, and the enhancement antigen presenting cells is offered antigenic processing, increases the output of specific antibody, can induce the Th1 para-immunity to reply under IL-12 and IFN-γ effect, produces stronger cellular immunization.Mouse, CpG-ODN has been used for hen's egg-white lysozyme, Protalbinic acid, Toxoid,tetanus, hepatitis B surface antigen, inactivating influenza virus, multiple vaccine and the antigenic researchs of model such as protein binding polysaccharide and dna vaccination, can both obviously strengthen humoral immunization and cellular immunization, and make immunne response deflection Th1 direction.Unite and use CpG-ODN and Al (OH)
3As the recombinant hepatitis b vaccine adjuvant, not only make the surface antigen antibody titre increase by 35 times, but also removed the restraining effect of aluminium adjuvant pair cell immunity, produced cellular immunization at surface antigen
[5]
Therefore reply owing to CpG-ODN induces Th1 to suppress the Th2 para-immunity, can be used for treating the allergic diseases that the Th2 para-immunity is replied mediation, as asthma etc.In addition,, induce the various kinds of cell factor, have non-specific immune enhancing function, therefore can also be used to treating tumour, infectious diseases and immunologic hypofunction or defective because CpG-ODN can activate the panimmunity cell.
Dna vaccination has been obtained good immune effect in mouse, one of its mechanism is that CpG-ODN has brought into play important adjuvant effect in dna immunization, but same dna vaccination ubiquity DNA consumption in human body is big, the problem that immune response is weak, does this exist species variation relevant with the immunostimulatory activity of CpG-ODN? is there the CpG-ODN that people's immunocyte is had the specific immune stimulating activity? never have system's report in the world about this respect, but fragmentary data shows, some oligonucleotide also have stimulating activity to people's immunocyte, and not homotactic activity difference to some extent, may there be human specific CpG-ODN sequence in prompting.If there is human specific CpG-ODN really, may have potential application foreground to prevention and treatment of diseases such as human infectious disease, anaphylaxis, immune deficiency and tumours so.Therefore, whether exploration exists human specific CpG-ODN and find out its characteristic sequence just becomes main purpose of the present invention.
We at first design synthesized tens contain 5 '-CG-3 ' dinucleotides, length is the sulfo-CpG-ODN of 12 bases, and utilize the human peripheral blood single nucleus cell (PBMC) of vitro culture to set up screening system, found a plurality of hexabasic basic characteristic sequences that can activate human B cell: wherein four characteristic sequences have the immunostimulatory activity of moderate to human B cell, sequence is that 5 '-GACGTT-3 ', 5 '-GACGTC-3 ', 5 '-GGCGTT-3 ' and 5 '-GGCGTC-3 ' and the mouse characteristic sequence delivered are in full accord; And two characteristic sequences have immunostimulatory activity highly to human B cell in addition, and sequence is 5 '-GTCGTC-3 ' and 5 '-GTCGTT-3 ', and second bit base is " T ", and this bit base is " G " or " A " in the mouse characteristic sequence.According to latter two characteristic sequence, we design and have synthesized one group to contain 2～6 copy characteristic sequences, length be the CpG-ODN of 15～30 bases, have obtained a plurality of CpG-ODN that people's immunocyte had strong immunostimulatory activity through screening.We also find to exist the two class features human specific CpG-ODN of difference to some extent in experimentation: a class contains 5 '-GTCGTC-3 ' characteristic sequence, the immunostimulatory activity that people's immunocyte is had height, and mouse boosting cell is not had immunostimulatory activity, have the human specific of height; Another kind of 5 '-GTCGTT-3 ' characteristic sequence that contains has immunostimulatory activity preferably to people's immunocyte, and mouse boosting cell is also had the cross immunity stimulating activity.In sum, we have found that two have the CpG characteristic sequence of hyperimmunization stimulating activity and obtained a plurality of CpG-ODN that people's immunocyte had strong immunostimulatory activity people's immunocyte.
The present invention is described in detail below in conjunction with Figure of description: example 1 screening has the CpG-ODN characteristic sequence of immunostimulatory activity to human B cell
The CpG-ODN:5 '-X with following sequence has been synthesized in design
1X
2CGX
3X
4X
1X
2CGX
3X
4-3 ', X
iRepresent in A, G, four kinds of bases of C, T any, partial sequence is shown in table 1 (A).All sequences is synthetic after full chain thio-modification and PAGE purifying are made into 20 μ M with stroke-physiological saline solution.
Get healthy human peripheral blood under the aseptic condition, separate PBMC with lymphocyte separation medium, adjusting cell concn is 10
6/ ml inoculates 96 porocyte culture plates, every hole 200 μ l, add above-mentioned CpG-ODN to final concentration be 2 μ M, 37 ℃/5%CO
2Cultivated 8 days under the condition, with the IgM content in the double-antibody sandwich elisa method detection supernatant.
Have the CpG-ODN of the immunostimulatory activity B cell in can activating PBMC, proliferation and differentiation plasmablast behind the B cell activation, plasmocyte can produce immunoglobulin (Ig), is mainly IgM.Therefore detect the IgM level in the culture supernatant, just can assess the power of CpG-ODN people's immunocyte immunostimulatory activity.According to the IgM level, the immunostimulatory activity of CpG-ODN (2444 and 2443) that contains 5 '-GTCGTT-3 ' and 5 '-GTCGTC-3 ' characteristic sequence is the highest, the CpG-ODN (2144 that contains 5 '-GACGTT-3 ', 5 '-GACGTC-3 ', 5 '-GGCGTT-3 ' and 5 '-GGCGTC-3 ' characteristic sequence, 2143,2244 and 2243) have the immunostimulatory activity of moderate, other sequence does not have tangible active (see figure 1) to human B cell.2 pairs of human B cells of example have design and active detection of the CpG-ODN of immunostimulatory activity
The number of hexabasic basic characteristic sequence and array mode may influence the power of its immunostimulatory activity among the CpG-ODN, therefore we design again that to have synthesized a group length be 15～30 bases, contain the CpG-ODN of 2～6 copy characteristic sequences, and partial sequence is shown in table 1 (B).All sequences is synthetic after full chain thio-modification and PAGE purifying are made into 20 μ M with stroke-physiological saline solution.
Get healthy human peripheral blood under the aseptic condition, separate PBMC with lymphocyte separation medium, adjusting cell concn is 10
6/ ml inoculates 96 porocyte culture plates, every hole 200 μ l, add above-mentioned CpG-ODN to final concentration be 4,8,16,32 and 64nM, 37 ℃/5%CO
2Cultivated 8 days under the condition, with the IgM content in the double-antibody sandwich elisa method detection supernatant.
The result shows, the immunostimulatory activity of CpG-ODN (T7 and T8) that contains 5 or 6 copy 5 '-GTCGTC-3 ' characteristic sequences is the strongest, and other CpG-ODN also has activity; The activity of T7 and T8 is far above CpG-ODN (44444) (see figure 2) that only contains 3 copy same characteristic features sequences.The number of these presentation of results characteristic sequences and array mode influence the power of CpG-ODN to people's immunocyte immunostimulatory activity.Example 3 chloroquines are to the specific inhibitory effect of human specific CpG-ODN immunostimulatory activity
Proved that in mouse test chloroquine can specificity suppresses the immunostimulatory activity of CpG-ODN.In order to verify that CpG-ODN is special to the immunostimulatory activity of human B cell but not other factors causes, we have carried out following test:
Get healthy human peripheral blood under the aseptic condition, separate PBMC with lymphocyte separation medium, adjusting cell concn is 10
6/ ml inoculates 96 porocyte culture plates, every hole 200 μ l, add CpG-ODN to final concentration be 32nM, add chloroquine to final concentration again and be 0,10,100,1000 and 10000nM, 37 ℃/5%CO
2Cultivated 8 days under the condition, with the IgM content in the double-antibody sandwich elisa method detection supernatant.
Along with the increase of chloroquine concentration, the immunostimulatory activity of CpG-ODN is suppressed gradually, and the chloroquine of 1000nM has suppressed the active (see figure 3) of CpG-ODN fully, shows that CpG-ODN is special to the immunostimulatory activity of human B cell.Example 4 human specific CpG-ODN are to the influence of mouse boosting cell propagation
Get 10 all left and right sides Balb/c mouse, the execution of craning one, 75% ethanol disinfection, aseptic condition take out spleen down, push gently to obtain splenocyte with the elbow tweezers, separate spleen mononuclearcell (SMNC) with lymphocyte separation medium, and transferring cell concn is 10
6/ ml, inoculation 35mm Tissue Culture Dish, every ware 2ml, add CpG-ODN to final concentration be 100nM, 37 ℃/5%CO
2Cultivated 3 days under the condition, with the variation of microscope observing cell form, and the counting cells number.
SMNC is after mouse specific C pG-ODN (1826) stimulates, and cell volume increases, and cell number increases; But human specific CpG-ODN (D1, D2, D3, T7, T8 and 1826m) is not obvious to mouse SMNC effect, and only a few cell volume increases, and cell number and contrast do not have obviously difference (seeing Fig. 4 and Fig. 5).Example 5 human specific CpG-ODN are to the immunostimulatory activity of mouse spleen B cell
Get 10 all left and right sides Balb/c mouse, the execution of craning one, 75% ethanol disinfection, aseptic condition take out spleen down, push gently to obtain splenocyte with the elbow tweezers, separate SMNC with lymphocyte separation medium, and transferring cell concn is 10
6/ ml inoculates 96 porocyte culture plates, every hole 200 μ l, add CpG-ODN to final concentration be 4,8,16,32 and 64nM, 37 ℃/5%CO
2Cultivated 3 days under the condition, with the IgM content in the double-antibody sandwich elisa method detection supernatant.
Mouse specific C pG-ODN (1826) can activate mouse B cell, induces high-caliber IgM; But human specific CpG-ODN (D1, D2, D3, T7, T8 and 1826m) has only the immunostimulatory activity of moderate even non-activity (see figure 6) to mouse B cell.Comprehensive example 4 and example 5 results, human specific CpG-ODN has the human specific of height really.Reference 1.Tokunaga et al.Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG.1.isolation, physicochemical characterization, and antitumor activity J.Natl.Cancer Inst.1984,72:9552.Branda R.F.et al.Immune stimulation by an antisense oligomer which is complementary to the rev gene of HIV-1, Biochem.Pharmacol.1993,45:20373.Krieg AM.et al.CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995,374:5464.Sato Y.et al.Immunostimulatory DNA sequences necessary for effective intradermal gene immunization, Science 1996,273:3525.Davis HL.et al.CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen, J.Immunol. 1998,160:870
Claims (5)
1. the synthetic oligodeoxynucleotide that people's immunocyte is had the specific immune stimulating activity, its characteristics are for containing 5 '-GTCGTC-3 ' and/or 5 '-GTCGTT-3 ' characteristic sequence.
2. according to claim 1, characteristic sequence 5 '-GTCGTC-3 ' and 5 '-GTCGTT-3 ' is non-methylating.
3. according to claim 1, oligodeoxynucleotide is full chain thio-modification.
4. according to claim 1, oligodeoxynucleotide length is 10～40 bases.
5. the oligodeoxynucleotide that uses claim 1 to require prevents and treats the method for diseases such as human infectious disease, anaphylaxis, immune deficiency and tumour.
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| CN1296378C (en) * | 2004-05-17 | 2007-01-24 | 中国人民解放军第三军医大学 | CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof |
| WO2007012285A1 (en) * | 2005-07-28 | 2007-02-01 | Changchun Huapu Biotechnology Co., Ltd. | Viral infection resistent single strand deoxynucleosides |
| CN1307196C (en) * | 2004-01-08 | 2007-03-28 | 延世大学校 | Modified CpG oligodeoxynucleotide with improved immunoregulatory function |
| CN100439386C (en) * | 2003-03-05 | 2008-12-03 | 长春华普生物技术有限公司 | Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine |
| CN101492672B (en) * | 2008-01-23 | 2011-01-26 | 许洪林 | Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof |
| CN101148467B (en) * | 2006-09-20 | 2012-03-14 | 长春华普生物技术有限公司 | Oligonucleotide and use thereof for treating lung cancer |
| CN101559224B (en) * | 2008-04-18 | 2012-07-11 | 北京生物制品研究所 | Poliomyelitis vaccine |
| CN1688192B (en) * | 2002-08-19 | 2012-07-18 | 科勒制药集团有限公司 | Immunostimulatory nucleic acids |
| CN101675994B (en) * | 2008-09-19 | 2012-07-25 | 齐鲁制药有限公司 | Therapy vaccine preparation |
| CN101979566B (en) * | 2008-01-23 | 2013-05-01 | 许洪林 | Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof |
| WO2014001422A3 (en) * | 2012-06-28 | 2014-02-27 | Intervet International B.V. | Toll-like receptors and immunostimulatory oligonucleotides |
| CN102864152B (en) * | 2005-11-07 | 2015-11-18 | 艾德拉药物股份有限公司 | Comprise the immunostimulatory properties of the compound based on oligonucleotide of modified immunostimulating dinucleotides |
| CN112322626A (en) * | 2020-10-26 | 2021-02-05 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
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| CN1688192B (en) * | 2002-08-19 | 2012-07-18 | 科勒制药集团有限公司 | Immunostimulatory nucleic acids |
| CN100439386C (en) * | 2003-03-05 | 2008-12-03 | 长春华普生物技术有限公司 | Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine |
| CN1307196C (en) * | 2004-01-08 | 2007-03-28 | 延世大学校 | Modified CpG oligodeoxynucleotide with improved immunoregulatory function |
| CN1296378C (en) * | 2004-05-17 | 2007-01-24 | 中国人民解放军第三军医大学 | CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof |
| WO2007012285A1 (en) * | 2005-07-28 | 2007-02-01 | Changchun Huapu Biotechnology Co., Ltd. | Viral infection resistent single strand deoxynucleosides |
| CN102864152B (en) * | 2005-11-07 | 2015-11-18 | 艾德拉药物股份有限公司 | Comprise the immunostimulatory properties of the compound based on oligonucleotide of modified immunostimulating dinucleotides |
| CN101148467B (en) * | 2006-09-20 | 2012-03-14 | 长春华普生物技术有限公司 | Oligonucleotide and use thereof for treating lung cancer |
| CN101492672B (en) * | 2008-01-23 | 2011-01-26 | 许洪林 | Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof |
| CN101979566B (en) * | 2008-01-23 | 2013-05-01 | 许洪林 | Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof |
| CN101559224B (en) * | 2008-04-18 | 2012-07-11 | 北京生物制品研究所 | Poliomyelitis vaccine |
| CN101675994B (en) * | 2008-09-19 | 2012-07-25 | 齐鲁制药有限公司 | Therapy vaccine preparation |
| CN104718221A (en) * | 2012-06-28 | 2015-06-17 | 英特维特国际股份有限公司 | Toll-like receptors and immunostimulatory oligonucleotides |
| US20150191523A1 (en) * | 2012-06-28 | 2015-07-09 | Intervet Inc. | Toll-like receptors |
| JP2015522266A (en) * | 2012-06-28 | 2015-08-06 | インターベット インターナショナル ベー． フェー． | TOLL-like receptor |
| WO2014001422A3 (en) * | 2012-06-28 | 2014-02-27 | Intervet International B.V. | Toll-like receptors and immunostimulatory oligonucleotides |
| CN104718221B (en) * | 2012-06-28 | 2018-06-26 | 英特维特国际股份有限公司 | Toll-like receptor and immunostimulatory oligonucleotide |
| US10112985B2 (en) | 2012-06-28 | 2018-10-30 | Intervet Inc. | Toll-like receptors |
| CN112322626A (en) * | 2020-10-26 | 2021-02-05 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112322626B (en) * | 2020-10-26 | 2021-06-15 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
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