新型免疫佐剂CpG寡脱氧核苷酸对猪免疫学作用的研究
|
摘要
CpG基序是以未甲基化CpG二核苷酸为核心的寡聚脱氧核苷酸，其结构特征为：5’嘌呤-嘌呤-CpG-嘧啶-嘧啶3’。实验证明，人工合成的含CpG基序的寡脱氧核苷酸(CpG ODN)可以模拟细菌、病毒的自然危险信号，诱发机体产生强烈的Th1型免疫应答，有望成为一类新型的免疫佐剂。进一步的研究表明，哺乳动物对CpG ODN的识别具有种属特异性。本实验致力于研究体外对猪免疫细胞具有刺激活性的CpG ODN的结构特点，为进一步研究适用于猪的疫苗佐剂提供实验参考。
依据相关资料，设计并合成了38条CpG ODN。应用淋巴细胞增殖实验(LPR)和IFN-γ分泌实验来评价体外CpG ODN对猪外周血单个核细胞(PBMC)的免疫刺激活性。采用外周血单个核细胞培养、[~3H]-脱氧胸苷掺入检测技术，以刺激指数(SI值)为检测指标，比较了不同的CpG ODN刺激猪PBMC增殖的作用。发现具有免疫刺激作用的CpG ODN具有如下结构特点：①CpG ODN的作用依赖于序列中CpG二核苷酸的存在；②长度在20～25bp、含有三个或四个基序为最适序列；③ODN序列的磷酸戊糖骨架修饰对其作用也有影响。采用双抗体夹心ELISA检测了PBMC培养上清液中的IFN-γ的含量，发现能有效刺激猪PBMC分泌IFN-γ的序列是：以“嘌呤-嘧啶-CG-嘌呤-嘧啶”基序为中心，有一定的侧翼序列，并形成一种互补的回文结构；在3’端和5’端还加有poly G尾；为磷酸二酯键(PO)／硫代磷酸二酯键(PTO)混合型骨架，中心的碱基由磷酸二酯键连接，两端的poly G尾由硫代磷酸二酯键连接。在研究中还发现，能刺激猪PBMC增殖的CpG ODN和刺激其分泌IFN-γ的CpG ODN为两类结构不同的ODN。该实验结果为进一步研究适用于不同病原体的猪用疫苗佐剂奠定了基础。
CpG motif is a 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5'purines and two 3'pyrimidines. Synthetic Oligodeoxynucleotides (ODN) containing CpG motif were demonstrated to mimic a danger signal of bacterial or viral DNA and induce a potent Th1 biased immune response. Therefore, CpG ODN may have potential as a new adjuvant. Further studies documented that different motifs are optimally stimulatory in different species. The current work examines the structure character of the ODN which can stimulate porcine immune cells in vitro and provides an experimental foundation for intensive study on vaccine adjuvant for swine.
Using lymphocyte proliferative response (LPR) and IFN- Y secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on porcine peripheral blood mononuclear cells (PBMC) . The PBMC were cultured with ODN and the stimulation index was determined by 3H-TdR incorporation assay. Results show that the immunostimulatory CpG ODN has the following structure: (1)The CpG dinucleotide is needed; (2)The oligonucleotide is optimal which contains 3 or 4 motifs and whose length is between 20-25bp; (3)The modification of the backbone of the CpG ODN has a prominent influence on its immunostimulatory activity. The amount of IFN-γ in the culture supernatant was measured by swIFN-γ kit which is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) . This work demonstrates that the ODN which can stimulate porcine PBMC to secrete IFN- γ has the following structure : It has mixed phosphodiester/phosphorothioate backbone and contains a single hexameric purine/pyrimidine/CG/purine/pyrimidine motif flanked by self-complementary bases that form a stem-loop structure capped at the 3' and 5' end by a polyG tail. These findings indicate that porcine PBMC recognize and respond to two structurally distinct classes of CpG ODN. One class preferentially induces cell proliferation, whereas the other stimulates IFN- γ release. The distinct immunostimulatory properties of the two classes of CpG ODN improve the design of CpG ODN as vaccine adjuvants.
依据相关资料，设计并合成了38条CpG ODN。应用淋巴细胞增殖实验(LPR)和IFN-γ分泌实验来评价体外CpG ODN对猪外周血单个核细胞(PBMC)的免疫刺激活性。采用外周血单个核细胞培养、[~3H]-脱氧胸苷掺入检测技术，以刺激指数(SI值)为检测指标，比较了不同的CpG ODN刺激猪PBMC增殖的作用。发现具有免疫刺激作用的CpG ODN具有如下结构特点：①CpG ODN的作用依赖于序列中CpG二核苷酸的存在；②长度在20～25bp、含有三个或四个基序为最适序列；③ODN序列的磷酸戊糖骨架修饰对其作用也有影响。采用双抗体夹心ELISA检测了PBMC培养上清液中的IFN-γ的含量，发现能有效刺激猪PBMC分泌IFN-γ的序列是：以“嘌呤-嘧啶-CG-嘌呤-嘧啶”基序为中心，有一定的侧翼序列，并形成一种互补的回文结构；在3’端和5’端还加有poly G尾；为磷酸二酯键(PO)／硫代磷酸二酯键(PTO)混合型骨架，中心的碱基由磷酸二酯键连接，两端的poly G尾由硫代磷酸二酯键连接。在研究中还发现，能刺激猪PBMC增殖的CpG ODN和刺激其分泌IFN-γ的CpG ODN为两类结构不同的ODN。该实验结果为进一步研究适用于不同病原体的猪用疫苗佐剂奠定了基础。
CpG motif is a 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5'purines and two 3'pyrimidines. Synthetic Oligodeoxynucleotides (ODN) containing CpG motif were demonstrated to mimic a danger signal of bacterial or viral DNA and induce a potent Th1 biased immune response. Therefore, CpG ODN may have potential as a new adjuvant. Further studies documented that different motifs are optimally stimulatory in different species. The current work examines the structure character of the ODN which can stimulate porcine immune cells in vitro and provides an experimental foundation for intensive study on vaccine adjuvant for swine.
Using lymphocyte proliferative response (LPR) and IFN- Y secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on porcine peripheral blood mononuclear cells (PBMC) . The PBMC were cultured with ODN and the stimulation index was determined by 3H-TdR incorporation assay. Results show that the immunostimulatory CpG ODN has the following structure: (1)The CpG dinucleotide is needed; (2)The oligonucleotide is optimal which contains 3 or 4 motifs and whose length is between 20-25bp; (3)The modification of the backbone of the CpG ODN has a prominent influence on its immunostimulatory activity. The amount of IFN-γ in the culture supernatant was measured by swIFN-γ kit which is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) . This work demonstrates that the ODN which can stimulate porcine PBMC to secrete IFN- γ has the following structure : It has mixed phosphodiester/phosphorothioate backbone and contains a single hexameric purine/pyrimidine/CG/purine/pyrimidine motif flanked by self-complementary bases that form a stem-loop structure capped at the 3' and 5' end by a polyG tail. These findings indicate that porcine PBMC recognize and respond to two structurally distinct classes of CpG ODN. One class preferentially induces cell proliferation, whereas the other stimulates IFN- γ release. The distinct immunostimulatory properties of the two classes of CpG ODN improve the design of CpG ODN as vaccine adjuvants.
引文
1．张涛．Thl佐剂的合理设计．[J]．《国外医学》预防、诊断、治疗用生物制品分册，2002，25(2)：72-75
2．管孝鞠，吴玉章．Thl极化佐剂的研究进展．[J]．华西药学杂志，2001，16(2)：118-120
3. Wiemann, B, et al. Cotey's toxin, tumor necrosis factor and cancer research: a historical perspective.[J]. Pharmacol. Ther., 1994, 64: 529-564
4. Tokunaga T, Yamamoto H, Shimada S, et al. [J]. J Natl Cancer Inst, 1984, 72(4): 955-962
5. Tokunaga T, et al. How BCG led to the discovery of immunostimulatory DNA.[J] Jpn. J. Infect. Dis. 1999, 52: 1-11
6. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. [J]. Nature, 1995,374(6):546-549
7. Sato Y, Roman M, Tighe H, et al. [J]. Science, 1996,273(5273):352-354
8. Yi AK, Klinman DM, Martin TL, et al. Rapid immune activation by CpG motifs in bacterial DNA.Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. [J]. J. Immunol, 1996,157: 5394
9. Yi AK, Chang M, Peckham DW, et al. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry [J]. J. Immunol, 1998, 160: 5898-5906
10. Jakob T, Walker PS, Krieg AM, et al. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 response by immunostimulatory DNA. [J]. J. Immunol, 1998, 161, 3042-3049
11. Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA[J]. J. Immunol, 1996, 157: 1840
12. Cowdery JS, Chace JH, Yi AK, et al. Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides[J]. J. Immunol, 1996, 156: 4570
13. Bird AP.[J].Trends Genet,1987(3):342-347
14. Krieg AM, et al. Mechanism of action of CpG DNA.[J]. Curr, Top. Microbiol. Immunol., 2000,247:1-21
15. Takeshita F, Leifer CA, Gursel I, et al. Cutting edge:Role of Toll-Like Receptor 9 in CpG DNA-induced activation of human cells. [J]. J Immunol, 2001,167(7): 3555-3558
16. Bandholtz L, Guo Y, Palmberg C, et al. [J]. Cell Mol Life Sci, 2003,60(2): 422-429
17. Sun S, Beard C, Jaenisch R, et al. Mitogenicity of DNA from different organisms for murine B cells. [J]. J Immunol,1997, 159:3119-3125
18. Yamamoto S, Yamamoto T, Kataoka T, et al. [J]. Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity. J Immunol, 1992,148:4072-4076
19. Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune response in vitro and in vivo. [J]. J Immunol, 2000,164(3): 1617-1624
20. Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. [J]. J Immunol, 2000,164:944-952
21. Kamstrup S, Verthelyi D, Klinman DM. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides [J]. Vet Microbiol, 2001,78(4):353-362
22. Zhang Y, Shoda LM, Brayton KA, et al. [J]. J Interferon Cytokine Res, 2001,21(10):871-881
23. Dalpke AH, Zimmermann S, Albrecht I, et al. Phosphodiester CpG oligonucleotides as adjuvanta: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo.[J]. Immunol, 2002,106(1):102-112
24. Krieg AM, Wu T, Weeratna R, et al. [J]. Proc Natl Acad Sci U S A, 1998,95(21):12631-12636
25. Verthelyi D, Ishii KJ, Gursel M, et al. Humman peripheral blood cells differentially recognize and respond to two distinct CpG motif.[J]. J. Immunol, 2001, 166(4):2372-2377
26. Krieg AM. From A to Z on CpG. [J]. Trends Immunol, 2002, 23(2): 64-65
27. Verthelyi D, Gursel M, Kenney RT, et al. CpG oligodeoxynucleotides protect normal and SIV-infected Macaques from Leishmania infection. [J]. J Immunol, 2003, 170(9):4717-4723
28. Weiner GJ. [J]. J. Leukoc. Biol, 2000,68(10):455-463
29. Krieg AM, Yi AK, Schorr J, et al. The role of CpG dinucleotides in DNA vaccine. [J]. Trends Microbiol, 1998,6:23-27
30．许洪林，邓瑶，王世峰等．人CpG寡脱氧核苷酸对质粒DNA免疫刺激活性的影响．[J]．中华医学杂志，2002，82(7)：492-495
31. DNA vaccine web site:http://www.genweb.com/Dnavax/dnavax.html
32. Kline JN. Effects of CpG DNA on Th1/Th2 balance in asthma. [J]. Curr Top Microbiol Immunol, 1999,247:in press
33 Micronnet I, Koening S, Speiser D, et al. CpG are efficient adjuvants for specific CTL aduction agajnst tumor antigan-derived peptide. [J]. J Immunol, 2002, 168:1212-1218 (?)63
35. Brown WC, Estes DM, Chantler SE, et al. DNA and CpG oligonucleotides derived from Babesia bovis are mitogenic for bovine B cells. [J]. Infect Immun, 1998, 66:5423-5432
36. Pontarollo RA, Rankin R, Babiuk LA, et al. Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. [J]. Vet Immunol Immunopathol, 2002, 84:43-59
37．朱立平、陈学清主编．免疫学常用实验方法．[M]．人民军医出版社，2000
38．鄂征主编．组织培养和分子细胞学技术．[M]．北京出版社，2001
39. Krieg AM. The role of CpG motif in innate immunity. [J]. Cur Opin Immunol, 2000,12:35-43
40. Krieg AM. Immune effects and mechanisms of action of CpG motif.[J]. Vaccine, 19,618-622
41. Krieg AM. Now I know my CpGs. [J]. Trends Microbiol, 2001, 9(6): 249-252
42. Verthelyi D, Kenney RT, Seder RA, et al. CpG oligodeoxynucleotides as vaccine adjuvants in primates. [J]. J Immunol, 2002, 168:1659-1663
43. Pontarlllo RA, Rankin R, Babiuk LA, et al. Monocyte are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. [J]. Vet Immunol Immunopathol, 2002, 84: 43-59
44. Rankin R, Pontarollo R, Ioannou X, et al. Identification of a stimulatory CpG motif for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. [J]. Antisense and Nucleic Acid Drug Development, 2001, 11: 333-340
45. Veit H, Simon R, Stefanie B, et al. Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides. [J]. J Immunol, 2002, 168:4531-4537
46．赵建平，吴玉章，万瑛等．含CpG寡聚脱氧核苷酸对肿瘤抗原肽P815AB的免疫佐剂效应研究．[J]．第三军医大学学报，2000，22(10)：954-957
47．郭爱珍，唐泰山，陆承平．细菌CpG DNA对犬免疫增强效果的研究．[J]．中国兽医科技，2001，31(12)：6-8
48．李文波，姚志强，周永兴等．佐剂CpG ODN对HBV基因疫苗诱导抗体产生的影响．[J]．第四军医大学学报，2000，21(7)：799-801
49．李光金，李颖杰，严维耀等．CpG DNA增强口蹄疫病毒DNA疫苗诱发豚鼠产生的免疫应答．[J]．科学通报，2001，46(6)：477-480
50. Krieg AM. [J]. J Gene Med, 1999,1(1):56-63
51. Miconnet I, Koenig S, Speiser D, et al. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. [J].J Immunol, 2002, 168:1212-1218
52．王明俊等．兽医生物制品学．[M]．中国农业出版社．1997
2．管孝鞠，吴玉章．Thl极化佐剂的研究进展．[J]．华西药学杂志，2001，16(2)：118-120
3. Wiemann, B, et al. Cotey's toxin, tumor necrosis factor and cancer research: a historical perspective.[J]. Pharmacol. Ther., 1994, 64: 529-564
4. Tokunaga T, Yamamoto H, Shimada S, et al. [J]. J Natl Cancer Inst, 1984, 72(4): 955-962
5. Tokunaga T, et al. How BCG led to the discovery of immunostimulatory DNA.[J] Jpn. J. Infect. Dis. 1999, 52: 1-11
6. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. [J]. Nature, 1995,374(6):546-549
7. Sato Y, Roman M, Tighe H, et al. [J]. Science, 1996,273(5273):352-354
8. Yi AK, Klinman DM, Martin TL, et al. Rapid immune activation by CpG motifs in bacterial DNA.Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. [J]. J. Immunol, 1996,157: 5394
9. Yi AK, Chang M, Peckham DW, et al. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry [J]. J. Immunol, 1998, 160: 5898-5906
10. Jakob T, Walker PS, Krieg AM, et al. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 response by immunostimulatory DNA. [J]. J. Immunol, 1998, 161, 3042-3049
11. Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA[J]. J. Immunol, 1996, 157: 1840
12. Cowdery JS, Chace JH, Yi AK, et al. Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides[J]. J. Immunol, 1996, 156: 4570
13. Bird AP.[J].Trends Genet,1987(3):342-347
14. Krieg AM, et al. Mechanism of action of CpG DNA.[J]. Curr, Top. Microbiol. Immunol., 2000,247:1-21
15. Takeshita F, Leifer CA, Gursel I, et al. Cutting edge:Role of Toll-Like Receptor 9 in CpG DNA-induced activation of human cells. [J]. J Immunol, 2001,167(7): 3555-3558
16. Bandholtz L, Guo Y, Palmberg C, et al. [J]. Cell Mol Life Sci, 2003,60(2): 422-429
17. Sun S, Beard C, Jaenisch R, et al. Mitogenicity of DNA from different organisms for murine B cells. [J]. J Immunol,1997, 159:3119-3125
18. Yamamoto S, Yamamoto T, Kataoka T, et al. [J]. Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity. J Immunol, 1992,148:4072-4076
19. Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune response in vitro and in vivo. [J]. J Immunol, 2000,164(3): 1617-1624
20. Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. [J]. J Immunol, 2000,164:944-952
21. Kamstrup S, Verthelyi D, Klinman DM. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides [J]. Vet Microbiol, 2001,78(4):353-362
22. Zhang Y, Shoda LM, Brayton KA, et al. [J]. J Interferon Cytokine Res, 2001,21(10):871-881
23. Dalpke AH, Zimmermann S, Albrecht I, et al. Phosphodiester CpG oligonucleotides as adjuvanta: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo.[J]. Immunol, 2002,106(1):102-112
24. Krieg AM, Wu T, Weeratna R, et al. [J]. Proc Natl Acad Sci U S A, 1998,95(21):12631-12636
25. Verthelyi D, Ishii KJ, Gursel M, et al. Humman peripheral blood cells differentially recognize and respond to two distinct CpG motif.[J]. J. Immunol, 2001, 166(4):2372-2377
26. Krieg AM. From A to Z on CpG. [J]. Trends Immunol, 2002, 23(2): 64-65
27. Verthelyi D, Gursel M, Kenney RT, et al. CpG oligodeoxynucleotides protect normal and SIV-infected Macaques from Leishmania infection. [J]. J Immunol, 2003, 170(9):4717-4723
28. Weiner GJ. [J]. J. Leukoc. Biol, 2000,68(10):455-463
29. Krieg AM, Yi AK, Schorr J, et al. The role of CpG dinucleotides in DNA vaccine. [J]. Trends Microbiol, 1998,6:23-27
30．许洪林，邓瑶，王世峰等．人CpG寡脱氧核苷酸对质粒DNA免疫刺激活性的影响．[J]．中华医学杂志，2002，82(7)：492-495
31. DNA vaccine web site:http://www.genweb.com/Dnavax/dnavax.html
32. Kline JN. Effects of CpG DNA on Th1/Th2 balance in asthma. [J]. Curr Top Microbiol Immunol, 1999,247:in press
33 Micronnet I, Koening S, Speiser D, et al. CpG are efficient adjuvants for specific CTL aduction agajnst tumor antigan-derived peptide. [J]. J Immunol, 2002, 168:1212-1218 (?)63
35. Brown WC, Estes DM, Chantler SE, et al. DNA and CpG oligonucleotides derived from Babesia bovis are mitogenic for bovine B cells. [J]. Infect Immun, 1998, 66:5423-5432
36. Pontarollo RA, Rankin R, Babiuk LA, et al. Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. [J]. Vet Immunol Immunopathol, 2002, 84:43-59
37．朱立平、陈学清主编．免疫学常用实验方法．[M]．人民军医出版社，2000
38．鄂征主编．组织培养和分子细胞学技术．[M]．北京出版社，2001
39. Krieg AM. The role of CpG motif in innate immunity. [J]. Cur Opin Immunol, 2000,12:35-43
40. Krieg AM. Immune effects and mechanisms of action of CpG motif.[J]. Vaccine, 19,618-622
41. Krieg AM. Now I know my CpGs. [J]. Trends Microbiol, 2001, 9(6): 249-252
42. Verthelyi D, Kenney RT, Seder RA, et al. CpG oligodeoxynucleotides as vaccine adjuvants in primates. [J]. J Immunol, 2002, 168:1659-1663
43. Pontarlllo RA, Rankin R, Babiuk LA, et al. Monocyte are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. [J]. Vet Immunol Immunopathol, 2002, 84: 43-59
44. Rankin R, Pontarollo R, Ioannou X, et al. Identification of a stimulatory CpG motif for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. [J]. Antisense and Nucleic Acid Drug Development, 2001, 11: 333-340
45. Veit H, Simon R, Stefanie B, et al. Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides. [J]. J Immunol, 2002, 168:4531-4537
46．赵建平，吴玉章，万瑛等．含CpG寡聚脱氧核苷酸对肿瘤抗原肽P815AB的免疫佐剂效应研究．[J]．第三军医大学学报，2000，22(10)：954-957
47．郭爱珍，唐泰山，陆承平．细菌CpG DNA对犬免疫增强效果的研究．[J]．中国兽医科技，2001，31(12)：6-8
48．李文波，姚志强，周永兴等．佐剂CpG ODN对HBV基因疫苗诱导抗体产生的影响．[J]．第四军医大学学报，2000，21(7)：799-801
49．李光金，李颖杰，严维耀等．CpG DNA增强口蹄疫病毒DNA疫苗诱发豚鼠产生的免疫应答．[J]．科学通报，2001，46(6)：477-480
50. Krieg AM. [J]. J Gene Med, 1999,1(1):56-63
51. Miconnet I, Koenig S, Speiser D, et al. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. [J].J Immunol, 2002, 168:1212-1218
52．王明俊等．兽医生物制品学．[M]．中国农业出版社．1997