CN1810970B

CN1810970B - CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application

Info

Publication number: CN1810970B
Application number: CN2005100029570A
Authority: CN
Inventors: 王丽颖; 包木胜; 于永利
Original assignee: Changchun Huapu Biotechnology Co Ltd
Current assignee: Huapu Biotechnology Hebei Co ltd
Priority date: 2005-01-27
Filing date: 2005-01-27
Publication date: 2011-05-18
Anticipated expiration: 2025-01-27
Also published as: CN1810970A; WO2006079291A1

Abstract

The present invention relates to one kind of CpG-containing single-stranded deoxynucleotide capable of strengthening the immunostimulation of antigen or antigen composite, the vaccine composition of the single-stranded deoxynucleotide and antigen or antigen composite and its preparation process. The CpG-containing single-stranded deoxynucleotide can strengthen the specific antitumor, antiviral and chlamydia infection resisting activity of heat shock protein-antigen peptide fusion protein induced antigen peptide. In addition, the present invention also relates to the application of the CpG-containing single-stranded deoxynucleotide in preparing vaccine composition and the method of strengthening the immunostimulation. The vaccine composition may be used as treating medicine for viral infection, bacterial infectio, parasite infection, allergic reaction, cancer and relevant diseases.

Description

Contain single chain deoxynucleotide and its vaccine composition and the application thereof of CpG

Technical field

The present invention relates to a kind of can enhancement antigen or the single chain deoxynucleotide that contains CpGODN of antigen composition immunostimulation, and by described single chain deoxynucleotide and antigen or vaccine composition that antigen composition is formed.In addition, the invention still further relates to the application of single chain deoxynucleotide in the preparation vaccine composition that contains CpGODN, and described vaccine composition is as the application of medicine.

Background technology

1984, discovery bacille Calmette-Guerin vaccine (BCG) DNA such as Tokunaga T can suppress growth of tumor, activate human peripheral blood single nucleus cell and NK cell (NK) cell, inducement interferon (IFN), these functions depend on CpG structure (Tokunaga T in the dna molecular, Antitumor activity of deoxyribonucleic acid fractionfrom Mycobacterium bovis BCG.I.Isolation, physicochemicalcharacterization, and antitumor activity, JNCI, 72:955.1984).CpG is the dinucleotides that is connected into by phosphoric acid by cytosine(Cyt) and guanine., wherein C represents cytosine(Cyt), and G represents guanine, and p represents phosphoric acid, and cytosine(Cyt) is positioned at 5 ' end.Studies show that in recent years, the oligonucleotide single stranded DNA that contains one or more CpG (CpG ODN) of synthetic also can show immunoregulation effect, can strengthen the activity of B cell, T cell, NK cell, antigen presenting cell (as monocyte, scavenger cell and dendritic cell) and neutrophil leucocyte, and show tangible potential applicability in clinical practice (Weiner GJ, The immunobiology and clinicalpotential of immunostimulatory CpG oligodeoxynucleotides.J Leukoc Biol 2000Oct; 68 (4): 455-63).But because the difference of sequence, CpG ODN can have diversified form.

(heat shock protein is to be present in the intravital molecular chaperone protein matter family of multiple biology HSP) to heat shock protein(HSP).In the process of immunne response, heat shock protein(HSP) can show following four kinds of basic functions: 1, carry antigen and enter the antigen presenting cell that comprises dendritic cell; 2, guiding antigen to enter the processing of MHC I classpath in antigen presenting cell offers; 3, stimulate the antigen presenting cell that comprises dendritic cell to express collaborative stimulation molecule, secrete cytokines; 4, make the antigen that carries antigen and heat shock protein(HSP) formation mixture or fusion rotein, to obtain character (the Tamura Y of activation antigen specificity cell toxicity T lymphocyte, Peng P, LiuK, Daou M, Srivastava PK.Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science 1997 Oct3; 278 (5335): 117-20).

(cytotoxic T lymphocyte CTL) is an individual killing tumor cell the most powerful and the immunocyte that kills and wounds virus infected cell to cytotoxic T lymphocyte.Ectogenic protein antigen is after using individuality, in comprising the antigen presenting cell of dendritic cell, mainly offer through the processing of MHC II classpath, excite humoral immune reaction (Heikema A, Agsteribbe E, Wilschut J, Huckriede A.Generation ofheat shock protein-based vaccines by intracellular loading of gp96 withantigenic peptides.Immunol Lett 1997 Jun 1; 57 (1-3): 69-74), can not induce antigen-specific CTL effectively.

Mycobacterium heat shock protein 65 can carry with its fusion or bonded or link coupled antigen peptide enter MHC I class angtigen presentation approach, the cytotoxic T lymphocyte of activation antigen peptide specific kills and wounds the tumour cell of antigen expressed peptide.

Hepatitis C virus (HCV) is the pathogenic agent that causes hepatitis C, has infected about more than 100,007,000 ten thousand people in the whole world.Liver cancer and liver cirrhosis are that HCV infects two severe complications that cause.Worldwide, adopting alpha-interferon and virazole combined utilization is the method for treatment infection with hepatitis C virus, it is efficient to be about 40% (Jon Cohen.The Scientific Challenge of Hepatitis C.Science, 1999,285:26-30).Adopting tubercule bacillus heat shock protein(HSP) 65 (HSP65) and multi-epitope HCV cAg to merge formed fusion rotein mutually is a kind of genetically engineered recombinant protein vaccine (CN02122116.2, China) that hepatitis C is had prevention and therapeutic action.

Chlamydia trachomatis (Chlamydia Trachomatis, CT) be the endotrophic microorganism of franchise that does not move, can cause trachoma, inclusion conjunctivitis, genitourinary system, lymphogranuloma venereum, endometritis, salpingitis, pelvic inflammatory disease, tubal infertility and fallopian tube ectopic pregnancy the mankind.

The major outer membrane albumen (MOMP) of chlamydia trachomatis can stimulate protective immunity.Adopting tubercule bacillus heat shock protein(HSP) 65 and chlamydia trachomatis major outer membrane albumen epitope antigen peptide to merge formed fusion rotein mutually is a kind of recombinant protein (CN02141977.9, China) that people's chlamydia trachomatis infection and relative disease thereof is had prevention and therapeutic action.

(Prostate specific antigen PSA) is a kind of tissure specific antigen that is expressed in prostate cancer cell to human prostate-specific antigen.Adopting tubercule bacillus heat shock protein(HSP) 65 and PSA epitope antigen peptide to merge formed fusion rotein mutually is a kind of recombinant protein (CN01134935.2, China) that human prostata cancer is had prevention and therapeutic action.

MUC1 albumen is a kind of of Saliva Orthana (mucins), and high level expression is the target spot of immunotherapy at mammary cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer cell.Adopting tubercule bacillus heat shock protein(HSP) 65 and MUC1 antigen peptide to merge formed fusion rotein mutually is a kind of recombinant protein (CN01102614.6, China) that the expression proteic tumour of MUC1 (including but not limited to mammary cancer) is had prevention and therapeutic action.

HER-2 albumen (HER-2) is a kind of and EGF-R ELISA (Tadashi Yamamoto, et al.Similarity of protein encoded by the human c-erbB-2gene to epidermal growthfactor receptor.Nature vol 319 16 January 1986,230-234) has the transmembrane protein of very high homology, overexpression in HBT's cell of 30% is the target spot of mammary cancer immunotherapy.Adopting tubercule bacillus heat shock protein(HSP) 65 and HER-2 antigen peptide to merge formed fusion rotein mutually is a kind of recombinant protein (CN01136347.9, China) that human breast carcinoma is had prevention and therapeutic action.

Above-mentioned result of study shows, CpG ODN is had broad prospects with antigen or the antigen composition combined utilization that heat shock protein(HSP) merges mutually.Yet,, do not see relevant bibliographical information or open by till the present invention.

Summary of the invention

One of purpose of the present invention provides can enhancement antigen or the single chain deoxynucleotide that contains CpG of antigen composition immunostimulation, and its adjuvant that can be used as antigen or antigen composition comes the immunostimulation of enhancement antigen or antigen composition.They are made of the oligonucleotide single strand dna that contains one or more CpG, and its phosphodiester bond can be partial vulcanization, and all sulfurized also can be unvulcanized.

The single chain deoxynucleotide of the CpG of containing of the present invention comprises the sequence that is selected from following formula at least: (1) (G) _n(L) _nX ₁+ X ₂CGY ₁Y ₂(M) _n(G) _n, X wherein ₁Be A, T or G; X ₂Be A or T; Y ₁Be A or T; Y ₂Be A, T or C; L and M are respectively A, T, C or G; N is the integer of 0-6;

Preferably, the single chain deoxynucleotide that contains CpG has and is selected from following sequence shown in arbitrary:

5’-ggggTCgTTCgTCgTTgggggg-3’(SEQ ID NO：2)

5’-ggggATAACgTTgCgggggg-3’(SEQ ID NO：3)

5’-ggggTgCAACgTTCAgggggg-3’(SEQ ID NO：4)

5’-ggggTCCTACgTAggAgggggg-3’(SEQ ID NO：8)

5’-ggggTCCATgACgTTCCTgAAgggggg-3’(SEQ ID NO：23)

5’-gggggACgTCgCCggggggg-3’(SEQ ID NO：37)

5’-ggATCCgTACgCATgggggg-3’(SEQ ID NO：38)

5’-gggggAATCgATTCgggggg-3’(SEQ ID NO：101)

5’-gggATgCATCgATgCATCgggggg-3’(SEQ ID NO：100)

5’-ggTgCgACgTCgCAgggggg-3’(SEQ ID NO：102)

5’-gggACgTACgTCgggggg-3’(SEQ ID NO：105)

5’-gggggATCgACgTCgATCgggggg-3’(SEQ ID NO：108)

5’-ggCgATCgATCgATCggggggg-3’(SEQ ID NO：111)

5’-ggggTCgATCgATCgAgggggg-3’(SEQ ID NO：112)

5’-ggTCgCgATCgCgAgggggg-3’(SEQ ID NO：114)

5’-ggGGTCAACGTTGAgggggG-3’(SEQ ID NO：128)

5’-gTCgTTTTCgTCgACgAATTgggggggg-3’(SEQ ID NO：164)

5’-gTCgTTATCgTTTTTTCgTAgggggg-3’(SEQ ID NO：165)

5’-ggCgTTAACgACgggggg-3’(SEQ ID NO：166)

5’-gTCggCACgCgACgggggg-3’(SEQ ID NO：52)

5’-ggTgCgACgTCgCAgggggg-3’(SEQ ID NO：160)

5’-gTCTATTTTgTACgTACgTgggg-3’(SEQ ID NO：169)

5’-gACgTCgACgTCgACgTCAggggg-3’(SEQ ID NO：170)

5’-ggggTCgATCgTTgCTAgCgggggg-3’(SEQ ID NO：173)

5’-gggggACgTTATCgTATTggggggg-3’(SEQ ID NO：174)

5’-ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3’(SEQ ID NO：175)

5’-ACgATCgATCgATCgggggg-3’(SEQ ID NO：180)

5’-AgACgTCTAACgTCggggg-3’(SEQ ID NO：168)

5’-ggggTgCTggCCgTCgTTgggggg-3’(SEQ ID NO：5)

5’-ggggTCgTTgCCgTCgggggg-3’(SEQ ID NO：6)

5’-ACCggTATCgATgCCggTgggggg-3’(SEQ ID NO：22)

5’-TTCgTTgCATCgATgCATCgTTgggggg-3’(SEQ ID NO：28)

(2) (G) _n(L) _nCG (XY) _nCG (M) _n(G) _n, wherein X is A or T; Y is A or T; L and M are respectively A, T, C or G; N is the integer of 0-6;

The single chain deoxynucleotide that preferably contains CpG has and is selected from following sequence shown in arbitrary:

5’-ggggACgATACgTCggggggg-3’(SEQ ID NO：1)

5’-ggggACgATATCgATgggggg-3’(SEQ ID NO：7)

5’-ggACgATCgATCgTgggggg-3’(SEQ ID NO：99)

5’-TCggggACgATCgTCgggggg-3’(SEQ ID NO：106)

5’-gggggATCgATATCgATCgggggg-3’(SEQ ID NO：107)

5’-ggATCgATCgATCgATgggggg-3’(SEQ ID NO：110)

5’-ggTgCATCgATCgATgCAgggggg-3’(SEQ ID NO：109)

5’-ggTgCATCgTACgATgCAgggggg-3’(SEQ ID NO：104)

5’-ggTgCgATCgATCgCAgggggg-3’(SEQ ID NO：113)

5’-gggggggTCgATCgATgggggg-3’(SEQ ID NO：171)

5’-ggggTCgTCgAACgTTgggggg-3’(SEQ ID NO：172)

5’-TgTCgTTCCTTgTCgTT-3’(SEQ ID NO：16)

5’-TTCgCTTCgCTTTTCgCTTCgCTT-3’-3’(SEQ ID NO：29)

5’-ACCgCCAAggAgAAgCCgCAggAggg-3’(SEQ ID NO：44)

5’-TACAACggCgAggAATACC-3’(SEQ ID NO：46)

5’-gTACAACggCgAggAATACCT-3’(SEQ ID NO：48)

5’-ACCgTCgTTgCCgTCggCCC-3’(SEQ ID NO：49)

5’-TgCTggCCgTCgTT-3’(SEQ ID NO：50)

5’-gTCggCACgCgACg-3’(SEQ ID NO：51)

5’-gTCggCACgCgACgCCCCCC-3’(SEQ ID NO：53)

5’-TCCCgCTggACgTT-3’(SEQ ID NO：68)

5’-TTACCggTTAACgTTggCCggCC-3’(SEQ ID NO：75)

5’-ACCggTTAACgTTgTCCCCgggg-3’(SEQ ID NO：76)

5’-CgTTgACgATCgTCCCATggCggg-3’(SEQ ID NO：88)

5’-TCTgCggCCTTCgTCg-3’(SEQ ID NO：89)

5’-TAgTAACCggTCCggCgCCCCC-3’(SEQ ID NO：90)

5’-TTgCAgCgCTgCCggTggg-3’(SEQ ID NO：91)

5’-CggCCCATCgAgggCgACggC-3’(SEQ ID NO：93)

5’-TCATCgACTCTCgAgCgTTC-3’(SEQ ID NO：117)

5’-ATCgTCgACTCTCgTgTTCTC-3’(SEQ ID NO：118)

5’-TgCAgCTTgCTgCTTgCTgCTTC-3’(SEQ ID NO：127)

5’-ggTgCgACgTCgCAgATgAT-3’(SEQ ID NO：136)

5’-ggTCgAACgTTCgAgATgAT-3’(SEQ ID NO：137)

5’-gggggCgTCgTTTTCgTCgACgAATT-3’(SEQ ID NO：143)

5’-actcgagacgcccgttgatagctt-3’(SEQ ID NO：145)

5’-AACgTTggCgTCgACgTCAgCgCC-3’(SEQ ID NO：146)

5’-gACgTCgACgTTgACgCT-3’(SEQ ID NO：147)

5’-ggCgTTAACgTTAgCgCT-3’(SEQ ID NO：148)

5’-AgCgCTAgCgCTgACgTT-3’(SEQ ID NO：149)

5’-CTAgACgTTCAAgCgTT-3’(SEQ ID NO：150)

5’-gACgATCgTCgACgATCgTC-3’(SEQ ID NO：156)

5’-gTCgTTCgTAgTCgACTACgAgTT-3’(SEQ ID NO：161)

5’-AAAAgACgTCgACgTCgACgTCTTTT-3’(SEQ ID NO：162)

5’-TgCgACgATCgTCgCACgATCggAT-3’(SEQ ID NO：176)

5’-TgCgACgTCgCACAgCgT-3’(SEQ ID NO：177)

(3) (TCG) _n(L) _nCG (M) _n(G) _n, wherein L and M are respectively A, T, C or G; N is the integer of 0-6;

5’-TCgTTgCCgTCgg-3’(SEQ ID NO：59)

5’-TCgTTgCCgTCggg-3’(SEQ ID NO：60)

5’-TCgTTgCCgTCgggg-3’(SEQ ID NO：61)

5’-TCgTTgCCgTCggggg-3’(SEQ ID NO：62)

5’-TCgTTgCCgTCgggggg-3’(SEQ ID NO：63)

5’-TCgTTgCCgTCggggggg-3’(SEQ ID NO：64)

5’-TCgTTgCCgTCgggggggg-3’(SEQ ID NO：65)

5’-TCgTTgCCgTCggggggggg-3’(SEQ ID NO：66)

5’-TCgTCgggTgCATCgATgCAgggggg-3’(SEQ ID NO：103)

5’-TCgTCgggTgCAACgTTgCAgggggg-3’(SEQ ID NO：129)

5’-TCgTCgggTgCgTCgACgCAgggggg-3’(SEQ ID NO：130)

5’-TCgTCgggTgCgATCgCAgggggg-3’(SEQ ID NO：131)

5’-TCgTCgggTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：132)

5’-TCgTCgTgCgACgTCgCAgggggg-3’(SEQ ID NO：133)

5’-TCgTCgCAgAACgTTCTgggggg-3’(SEQ ID NO：134)

5’-TCgTgCgACgTCgCAgggggg-3’(SEQ ID NO：135)

5’-TCgTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：139)

5’-TCgTATgCATCgATgCATAgggAgg-3’(SEQ ID NO：140)

5’-TCgTgCATCgATgCAgggggg-3’(SEQ ID NO：157)

5’-TCgAAACgTTTCgggggg-3’(SEQ ID NO：158)

5’-TCggACgATCgTCgggggg-3’(SEQ ID NO：159)

5’-TCgAgCgATCgCTCgAgggggg-3’(SEQ ID NO：179)

5’-TCgTCgCTTTgTCgTTgggg-3’(SEQ ID NO：13)

5’-TCgTCgTTTTgTCgTTgggg-3’(SEQ ID NO：11)

5’-TCgTCgggTgCgACgTCgCAgggggg-3’(SEQ ID NO：18)

5’-TCgTCgggTgCgACgATCgTCgggggg-3’(SEQ ID NO：19)

5’-TCgTCgTTTgCATCgATgCAggggggg-3’(SEQ ID NO：21)

5’-TCgTCgTTTTgACgATCgTCgggggg-3’(SEQ ID NO：24)

5’-TCgTTCggggTgCCg-3’(SEQ ID NO：80)

5’-TCgTTCggggTACCgATgggg-3’(SEQ ID NO：84)

5’-TCgTTgCgCTCCCATgCCgggggg-3’(SEQ ID NO：85)

5’-TCgTCgTTTCgTCgTTgggg-3’(SEQ ID NO：86)

5’-TCgTTgTCgTTTCgCTgCCggCggggg-3’(SEQ ID NO：87)

5’-TgCTTgggTggCAgCTgCCAgggggg-3’(SEQ ID NO：125)

5’-TgCTgCTTTgCTgCTTgggg-3’(SEQ ID NO：126)

5’-AACgTTCgACgTCgAACggggggg-3’(SEQ ID NO：163)

5’-AACgACgACgTTggggg-3’(SEQ ID NO：167)

(4) (TCG) _n(L) _nX ₁X ₂CG (M) _n, X wherein ₁Be A, T or G; X ₂Be A or T; L and M are respectively A, T, C or G; N is the integer of 0-6;

5’-TCgTAACgTTgTTTTTAACgTT-3’(SEQ ID NO：9)

5’-TCgTCgTATACgACgATCgTT-3’(SEQ ID NO：10)

5’-TCgTCgTTTgCgTTgTCgTT-3’(SEQ ID NO：12)

5’-TCCTgTCgTTTTgTCgTT-3’(SEQ ID NO：14)

5’-TCgTCgTTgTCgTTCgCT-3’(SEQ ID NO：15)

5’-TCgTCgTTACCgATgACgTCgCCgT-3’(SEQ ID NO：17)

5’-TCgTCgTTTgCATCgATgCAgTCgTCgTT-3’(SEQ ID NO：20)

5’-TCgCCTCgTCgCCTTCgAgC-3’g-3’(SEQ ID NO：30)

5’-TCgTgTgCgTgCCgTTggg-3’T-3’(SEQ ID NO：31)

5’-TCgTCgAgggCgCCggTgAC-3’-3’(SEQ ID NO：32)

5’-TCgTCgCCggTgggggTgTg-3’3’(SEQ ID NO：33)

5’-TCgTCgTACgCAATTgTCTT-3’3’(SEQ ID NO：34)

5’-TCgCCCACCggTgggggggg-3’3’(SEQ ID NO：35)

5’-TCgTCgCAgACCggTCTgggg-3’(SEQ ID NO：36)

5’-TCgTCgCggCCggCgCCCCC-3’(SEQ ID NO：39)

5’-TCgTCgCggCCgCgAggggg-3’(SEQ ID NO：40)

5’-TCgAggACAAgATTCTCgTgC-3’(SEQ ID NO：41)

5’-TCgAggACAAgATTCTCgTgCAggCC-3’(SEQ ID NO：42)

5’-TCgTgCAggCCAACgAggCCg-3’(SEQ ID NO：43)

5’-TCgTTgCCgTCggCCC-3’(SEQ ID NO：45)

5’-TCggCACgCgACgTgCTggCCgTCgTTTCC-3’(SEQ ID NO：47)

5’-TCgTTgCCgTCggCCCCCCCCC-3’(SEQ ID NO：54)

5’-TCgTTgCCgTCggCCCCCC-3’(SEQ ID NO：55)

5’-TCgTTgCCgTCggCCCCC-3’(SEQ ID NO：56)

5’-TCgTTgCCgTCggCCCC-3’(SEQ ID NO：57)

5’-TCgTTgCCgTCggCCCCCCC-3’(SEQ ID NO：58)

5’-TCgAggACAAgATTCTCgT-3’(SEQ ID NO：67)

5’-TCggCACgCgACgTgCTggCCgTCgTT-3’(SEQ ID NO：69)

5’-TCgTCgCgCCgTCACgggggg-3’(SEQ ID NO：70)

5’-TCgTgTgCgTgCCgTTggg-3’(SEQ ID NO：71)

5’-TCgTCgCCgTTgggCggg-3’(SEQ ID NO：72)

5’-TCgTCgACgTCgTTgggCggg-3’(SEQ ID NO：73)

5’-TCgCAgTTgTCgTAACgTTgggCggg-3’(SEQ ID NO：74)

5’-TCgTCgTTggTATgTT-3’(SEQ ID NO：77)

5’-TCgTCgTCgTCgTTgTCgTT-3’(SEQ ID NO：78)

5’-TCgTCgTCgTCgTTgTCgTTgggg-3’(SEQ ID NO：79)

5’-TCgTTCggggTgCCg-3’(SEQ ID NO：80)

5’-TCgTTCggggTAACgATT-3’(SEQ ID NO：81)

5’-TCgTTCggggTAACgTT-3’(SEQ ID NO：82)

5’-TCgTTCggggTACCgAT-3’(SEQ ID NO：83)

5’-TCgTACggCCgCCgTACggCggg-3’(SEQ ID NO：92)

5’-TCgCgTCgACTCCCCTCgAgggg-3’(SEQ ID NO：94)

5’-TCgTCgTCgACTCgTggTCggggg-3’(SEQ ID NO：95)

5’-TCgggCgCCCgATCgggggg-3’(SEQ ID NO：96)

5’-TCgTCggTCTTTCgAAATT-3’(SEQ ID NO：97)

5’-TCgTgACgTCCTCgAgTT-3’(SEQ ID NO：98)

5’-TCgTCTTTCgACTCgTTCTC-3’(SEQ ID NO：115)

5’-TCgTCgTTTTgCgTTCTC-3’(SEQ ID NO：116)

5’-TCgACTTTCgTCgTTCTgTT-3’(SEQ ID NO：119)

5’-TCgTCgTTTCgTCgTTCTC-3’(SEQ ID NO：120)

5’-TCgTCgTCgTCgTTgTCgTT-3’(SEQ ID NO：121)

5’-TCgTTCTCgACTCgTTCTC-3’(SEQ ID NO：122)

5’-TCgACgTTCgTCgTTCgTCgTTC-3’(SEQ ID NO：123)

5’-TCgTCgACgTCgTTCgTTCTC-3’(SEQ ID NO：124)

5’-TCgTgCgACgTCgCAgATgAT-3’(SEQ ID NO：138)

5’-TCgTCgAgCgCTCgATCggAT-3’(SEQ ID NO：141)

5’-TCgTCgTTTCgTAgTCgTTgACgTCggg-3’(SEQ ID NO：142)

5’-TCgTCggACgTTTTCCgACgTTCT-3’(SEQ ID NO：144)

5’-TCgTCgTTTTCgTCgTTTTCgTCgTT-3’(SEQ ID NO：151)

5’-TCgTCgTTTgTCgTgTgTCgTT-3；(SEQ ID NO：152)

5’-TCgTCgTTggTCggggTCgTTggggTCgTT-3’(SEQ ID NO：153)

5’-TCgTCgTTTCgTCTCTCgTT-3’(SEQ ID NO：154)

5’-TCgTCgTTTTgCTgCgTCgTT-3’(SEQID NO：155)

5’-TCgAgCgTTTTCgCTCgAATT-3’(SEQ ID NO：178)

(5) comprise the sequence of TTCGTCG.

5’-TTCgTCgTTTgATCgATgTTCgTTgggggg-3’(SEQ ID NO：25)’

5’-TTCgTCgTTgTgATCgATgggggg-3’-3’(SEQ ID NO：26)

5’-TATCgATgTTTTCgTCgTCgTTgggggg-3’(SEQ ID NO：27)

5’-TCgACTTTCgTCgTTCTgTT-3’(SEQ ID NO：119)

5’-TCgTCgTTTCgTCgTTCTC-3’(SEQ ID NO：120)

5’-TCgACgTTCgTCgTTCgTCgTTC-3’(SEQ ID NO：123)

5’-TCgTCgTTTTCgTCgTTTTCgTCgTT-3’(SEQ ID NO：151)

The single chain deoxynucleotide of the CpG of containing of the present invention also comprises the modification of each group on the base in the single chain deoxynucleotide, wherein said modification comprises that non-thio-modification, thio-modification, part thio-modification, rare base modification (dI, dU etc.), the modification that methylates, sulfydryl, Aminolinker C6 or Thiol-C6 S-S etc. are used for and the applied modification mode of other material couplings.

Another object of the present invention provides the vaccine composition of being made up of above-mentioned single chain deoxynucleotide and antigen or antigen composition, wherein the preferred of antigen or antigen composition is mixture or the fusion rotein that is formed by mycobacterium heat shock protein 65 and antigen peptide, and this mixture or fusion rotein can induce the preparation of antigen-specific CTL.Described antigen peptide is meant to have immunogenic peptide section or form under mixture, coupled thing or the fusion rotein state at the protein with other to have immunogenic peptide section, for example: the multi-epitope core antigen of C type hepatitis virus antigen peptide among the CN02122116.2, the relative disease that this antigen peptide causes infection with hepatitis C virus and infection with hepatitis C virus has prevention and therapeutic action, and concrete sequence is as follows:

Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Glu Ile Asp

Asp Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro

Leu Glu Asp Ser Glu Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro

Arg Leu Gly Val Arg Ala Glu Asn Asp Glu Ile Glu Gly Pro Arg

Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Asn Asp Glu

Leu Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln

Glu Ile Asp Asn Glu

Chlamydia trachomatis among the CN02141977.9 is mainly the multi-epitope antigen polypeptide, the effect that it has treatment and prevent the relative disease that is caused by chlamydozoan and choamydiae infection, and concrete sequence is as follows:

Glu Phe Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu Met Phe Thr

Asn Ala Ala Cys Met Ala Leu Asn Ile Trp Asp Glu Leu Asn Val Leu

Cys Asn Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly

Lys Glu Phe Pro Leu Ala Leu Asp Ala Ala Thr Gly Thr Lys Asp Ala

Ser Ile Asp Tyr His Glu Trp Gln Ala Ser Leu Ala Leu Ser Tyr Arg

Leu Asn Met Phe Thr Pro Tyr Ile Gly Val Lys Trp Ser Arg Ala Ser

Phe Asp Ala Asp Thr Tyr Lys Leu

Single copy antigenic peptide of the human prostate-specific cell antigen toxic T lymphocyte multi-epitope among the CN01134935.2, its prostate cancer to the people has the effect of prevention and treatment, and concrete sequence is as follows:

Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser

Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys

MUC1 cell antigen toxic T lymphocyte epitope antigen peptide among the CN011026146, it includes but not limited to that to the tumour of expressing MUC1 mammary cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer cell etc. have the effect of prevention and treatment, and concrete sequence is as follows:

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp

Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val

Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu

Multi-epitope HER-2 antigen peptide among the CN011363479, it includes but not limited to that to the tumour of expressing HER-2 mammary cancer, ovarian cancer, cancer of the stomach, carcinoma of endometrium, salivary-gland carcinoma, gland cancer, preceding gland cancer, colon and the rectum cancer, nonsmall-cell lung cancer, adenocarcinoma of lung etc. have the effect of treatment and prevention, and concrete sequence is as follows:

Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala Asn Ile Gln

Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro

Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro

Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile

Another object of the present invention is to provide a kind of method of producing vaccine composition, comprise that the single chain deoxynucleotide that contains CpG with significant quantity mixes with the antigen or the antigen composition of significant quantity.Preferably the single chain deoxynucleotide that contains CpG with significant quantity mixes mutually with the mixture or the fusion rotein that are formed by mycobacterium heat shock protein 65 and antigen peptide of significant quantity.

Another aspect of the present invention provides the method for a kind of enhancement antigen or antigen composition immunostimulation, and it is to unite use with containing the single chain deoxynucleotide of CpG and antigen or antigen composition.

Result of study of the present invention shows, when the single chain deoxynucleotide that contains CpG mixes use with mixture that is formed by mycobacterium heat shock protein 65 and antigen peptide or fusion rotein, the single chain deoxynucleotide that contains CpG can significantly strengthen the activity that mycobacterium heat shock protein 65 antigen peptide fusion roteins induce the antigen peptide specific CTL, significantly strengthening mycobacterium heat shock protein 65 antigen peptide fusion roteins stimulates mouse to suppress the activity of antigen expressed peptide growth of tumour cell, and significantly strengthening mycobacterium heat shock protein 65 antigen peptide fusion roteins, to induce antigen peptide specific antitumor, the activity of antiviral and anti-choamydiae infection.

The vaccine composition that provides on the one hand more of the present invention is used for the treatment of application in the vaccine of virus infection, infectation of bacteria, parasitic infection, transformation reactions or cancer in preparation.Comprise and to grant the people or the Mammals of needs treatment by the immune composition that the single chain deoxynucleotide that contains CpG and antigen or antigen composition are formed.The relative disease that described virus infection, infectation of bacteria, parasitic infection, transformation reactions or cancer include but are not limited to: to be caused by infection with hepatitis C virus, the relative disease, prostate cancer, mammary cancer, ovarian cancer, lung cancer, cancer of the stomach, carcinoma of endometrium, salivary-gland carcinoma, gland cancer, colon and the rectum cancer that cause by chlamydozoan and choamydiae infection, nonsmall-cell lung cancer, adenocarcinoma of lung etc.

Embodiment

Below in conjunction with concrete preparation embodiment and biology effect embodiment, the present invention is described in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art, and for example synthetic employing solid phase phosphoramidite triester method, ELISA adopt indirect method.

In following embodiment, the source of agents useful for same, trade(brand)name and/or be necessary to list its moiety person are all only indicated once.Do not giving unnecessary details foregoing if no special instructions at used identical reagent thereafter.

The design of embodiment 1 CpG single chain deoxynucleotide

Implementation sequence is as follows:

(1)(G) _n(L) _nX ₁X ₂CGY ₁Y ₂(M) _n(G) _n

X ₁=A, T, G; X ₂=A, T; Y ₁=A, T; Y ₂=A, T, C; L, M=A, T, C, G; N is 0-6

5’-ggggTCgTTCgTCgTTgggggg-3’(SEQ ID NO：2)

5’-ggggATAACgTTgCgggggg-3’(SEQ ID NO：3)

5’-ggggTgCAACgTTCAgggggg-3’(SEQ ID NO：4)

5’-ggggTCCTACgTAggAgggggg-3’(SEQ ID NO：8)

5’-ggggTCCATgACgTTCCTgAAgggggg-3’(SEQ ID NO：23)

5’-gggggACgTCgCCggggggg-3’(SEQ ID NO：37)

5’-ggATCCgTACgCATgggggg-3’(SEQ ID NO：38)

5’-gggggAATCgATTCgggggg-3’(SEQ ID NO：101)

5’-gggATgCATCgATgCATCgggggg-3’(SEQ ID NO：100)

5’-ggTgCgACgTCgCAgggggg-3’(SEQ ID NO：102)

5’-gggACgTACgTCgggggg-3’(SEQ ID NO：105)

5’-gggggATCgACgTCgATCgggggg-3’(SEQ ID NO：108)

5’-ggCgATCgATCgATCggggggg-3’(SEQ ID NO：111)

5’-ggggTCgATCgATCgAgggggg-3’(SEQ ID NO：112)

5’-ggTCgCgATCgCgAgggggg-3’(SEQ ID NO：114)

5’-ggGGTCAACGTTGAgggggG-3’(SEQ ID NO：128)

5’-gTCgTTTTCgTCgACgAATTgggggggg-3’(SEQ ID NO：164)

5’-gTCgTTATCgTTTTTTCgTAgggggg-3’(SEQ ID NO：165)

5’-ggCgTTAACgACgggggg-3’(SEQ ID NO：166)

5’-gTCggCACgCgACgggggg-3’(SEQ ID NO：52)

5’-ggTgCgACgTCgCAgggggg-3’(SEQ ID NO：160)

5’-gTCTATTTTgTACgTACgTgggg-3’(SEQ ID NO：169)

5’-gACgTCgACgTCgACgTCAggggg-3’(SEQ ID NO：170)

5’-ggggTCgATCgTTgCTAgCgggggg-3’(SEQ ID NO：173)

5’-gggggACgTTATCgTATTggggggg-3’(SEQ ID NO：174)

5’-ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3’(SEQ ID NO：175)

5’-ACgATCgATCgATCgggggg-3’(SEQ ID NO：180)

5’-AgACgTCTAACgTCggggg-3’(SEQ ID NO：168)

5’-ggggTgCTggCCgTCgTTgggggg-3’(SEQ ID NO：5)

5’-ggggTCgTTgCCgTCgggggg-3’(SEQ ID NO：6)

5’-ACCggTATCgATgCCggTgggggg-3’(SEQ ID NO：22)

5’-TTCgTTgCATCgATgCATCgTTgggggg-3’(SEQ ID NO：28)

(2)(G) _n(L) _nCG(XY) _nCG(M) _n(G) _n

X=A, T; Y=A, T; L, M=A, T, C, G; N is 0-6

5’-ggggACgATACgTCggggggg-3’(SEQ ID NO：1)

5’-ggggACgATATCgATgggggg-3’(SEQ ID NO：7)

5’-ggACgATCgATCgTgggggg-3’(SEQ ID NO：99)

5’-TCggggACgATCgTCgggggg-3’(SEQ ID NO：106)

5’-gggggATCgATATCgATCgggggg-3’(SEQ ID NO：107)

5’-ggATCgATCgATCgATgggggg-3’(SEQ ID NO：110)

5’-ggTgCATCgATCgATgCAgggggg-3’(SEQ ID NO：109)

5’-ggTgCATCgTACgATgCAgggggg-3’(SEQ ID NO：104)

5’-ggTgCgATCgATCgCAgggggg-3’(SEQ ID NO：113)

5’-gggggggTCgATCgATgggggg-3’(SEQ ID NO：171)

5’-ggggTCgTCgAACgTTgggggg-3’(SEQ ID NO：172)

5’-TgTCgTTCCTTgTCgTT-3’(SEQ ID NO：16)

5’-TTCgCTTCgCTTTTCgCTTCgCTT-3’-3’(SEQ ID NO：29)

5’-ACCgCCAAggAgAAgCCgCAggAggg-3’(SEQ ID NO：44)

5’-TACAACggCgAggAATACC-3’(SEQ ID NO：46)

5’-gTACAACggCgAggAATACCT-3’(SEQ ID NO：48)

5’-ACCgTCgTTgCCgTCggCCC-3’(SEQ ID NO：49)

5’-TgCTggCCgTCgTT-3’(SEQ ID NO：50)

5’-gTCggCACgCgACg-3’(SEQ ID NO：51)

5’-gTCggCACgCgACgCCCCCC-3’(SEQ ID NO：53)

5’-TCCCgCTggACgTT-3’(SEQ ID NO：68

5’-TTACCggTTAACgTTggCCggCC-3’(SEQ ID NO：75)

5’-ACCggTTAACgTTgTCCCCgggg-3’(SEQ ID NO：76)

5’-CgTTgACgATCgTCCCATggCggg-3’(SEQ ID NO：88)

5’-TCTgCggCCTTCgTCg-3’(SEQ ID NO：89)

5’-TAgTAACCggTCCggCgCCCCC-3’(SEQ ID NO：90)

5’-TTgCAgCgCTgCCggTggg-3’(SEQ ID NO：91)

5’-CggCCCATCgAgggCgACggC-3’(SEQ ID NO：93)

5’-TCATCgACTCTCgAgCgTTC-3’(SEQ ID NO：117)

5’-ATCgTCgACTCTCgTgTTCTC-3’(SEQ ID NO：118)

5’-TgCAgCTTgCTgCTTgCTgCTTC-3’(SEQ ID NO：127)

5’-ggTgCgACgTCgCAgATgAT-3’(SEQ ID NO：136)

5’-ggTCgAACgTTCgAgATgAT-3’(SEQ ID NO：137)

5’-gggggCgTCgTTTTCgTCgACgAATT-3’(SEQ ID NO：143)

5’-actcgagacgcccgttgatagctt-3’(SEQ ID NO：145)

5’-AACgTTggCgTCgACgTCAgCgCC-3’(SEQ ID NO：146)

5’-gACgTCgACgTTgACgCT-3’(SEQ ID NO：147)

5’-ggCgTTAACgTTAgCgCT-3’(SEQ ID NO：148)

5’-AgCgCTAgCgCTgACgTT-3’(SEQ ID NO：149)

5’-CTAgACgTTCAAgCgTT-3’(SEQ ID NO：150)

5’-gACgATCgTCgACgATCgTC-3’(SEQ ID NO：156)

5’-gTCgTTCgTAgTCgACTACgAgTT-3’(SEQ ID NO：161)

5’-AAAAgACgTCgACgTCgACgTCTTTT-3’(SEQ ID NO：162)

5’-TgCgACgATCgTCgCACgATCggAT-3’(SEQ ID NO：176)

5’-TgCgACgTCgCACAgCgT-3’(SEQ ID NO：177)

(3)(TCG) _n(L) _nCG(M) _n(G) _n

L, M=A, T, C, G; N is 0-6

5’-TCgTTgCCgTCgg-3’(SEQ ID NO：59)

5’-TCgTTgCCgTCggg-3’(SEQ ID NO：60)

5’-TCgTTgCCgTCgggg-3’(SEQ ID NO：61)

5’-TCgTTgCCgTCggggg-3’(SEQ ID NO：62)

5’-TCgTTgCCgTCgggggg-3’(SEQ ID NO：63)

5’-TCgTTgCCgTCggggggg-3’(SEQ ID NO：64)

5’-TCgTTgCCgTCgggggggg-3’(SEQ ID NO：65)

5’-TCgTTgCCgTCggggggggg-3’(SEQ ID NO：66)

5’-TCgTCgggTgCATCgATgCAgggggg-3’(SEQ ID NO：103)

5’-TCgTCgggTgCAACgTTgCAgggggg-3’(SEQ ID NO：129)

5’-TCgTCgggTgCgTCgACgCAgggggg-3’(SEQ ID NO：130)

5’-TCgTCgggTgCgATCgCAgggggg-3’(SEQ ID NO：131)

5’-TCgTCgggTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：132)

5’-TCgTCgTgCgACgTCgCAgggggg-3’(SEQ ID NO：133)

5’-TCgTCgCAgAACgTTCTgggggg-3’(SEQ ID NO：134)

5’-TCgTgCgACgTCgCAgggggg-3’(SEQ ID NO：135)

5’-TCgTgCgACgATCgTCgCAgggggg-3’(SEQ ID NO：139)

5’-TCgTATgCATCgATgCATAgggAgg-3’(SEQ ID NO：140)

5’-TCgTgCATCgATgCAgggggg-3’(SEQ ID NO：157)

5’-TCgAAACgTTTCgggggg-3’(SEQ ID NO：158)

5’-TCggACgATCgTCgggggg-3’(SEQ ID NO：159)

5’-TCgAgCgATCgCTCgAgggggg-3’(SEQ ID NO：179)

5’-TCgTCgCTTTgTCgTTgggg-3’(SEQ ID NO：13)

5’-TCgTCgTTTTgTCgTTgggg-3’(SEQ ID NO：11)

5’-TCgTCgggTgCgACgTCgCAgggggg-3’(SEQ ID NO：18)

5’-TCgTCgggTgCgACgATCgTCgggggg-3’(SEQ ID NO：19)

5’-TCgTCgTTTgCATCgATgCAggggggg-3’(SEQ ID NO：21)

5’-TCgTCgTTTTgACgATCgTCgggggg-3’(SEQ ID NO：24)

5’-TCgTTCggggTgCCg-3’(SEQ ID NO：80)

5’-TCgTTCggggTACCgATgggg-3’(SEQ ID NO：84)

5’-TCgTTgCgCTCCCATgCCgggggg-3’(SEQ ID NO：85)

5’-TCgTCgTTTCgTCgTTgggg-3’(SEQ ID NO：86)

5’-TCgTTgTCgTTTCgCTgCCggCggggg-3’(SEQ ID NO：87)

5’-TgCTTgggTggCAgCTgCCAgggggg-3’(SEQ ID NO：125)

5’-TgCTgCTTTgCTgCTTgggg-3’(SEQ ID NO：126)

5’-AACgTTCgACgTCgAACggggggg-3’(SEQ ID NO：163)

5’-AACgACgACgTTggggg-3’(SEQ ID NO：167)

(4)(TCG) _n(L) _nX ₁X ₂CG (M) _n

X ₁=A, T, G; X ₂=A, T; L, M=A, T, C, G; N is 0-6;

5’-TCgTAACgTTgTTTTTAACgTT-3’(SEQ ID NO：9)

5’-TCgTCgTATACgACgATCgTT-3’(SEQ ID NO：10)

5’-TCgTCgTTTgCgTTgTCgTT-3’(SEQ ID NO：12)

5’-TCCTgTCgTTTTgTCgTT-3’(SEQ ID NO：14)

5’-TCgTCgTTgTCgTTCgCT-3’(SEQ ID NO：15)

5’-TCgTCgTTACCgATgACgTCgCCgT-3’(SEQ ID NO：17)

5’-TCgTCgTTTgCATCgATgCAgTCgTCgTT-3’(SEQ ID NO：20)

5’-TCgCCTCgTCgCCTTCgAgC-3’g-3’(SEQ ID NO：30)

5’-TCgTgTgCgTgCCgTTggg-3’T-3’(SEQ ID NO：31)

5’-TCgTCgAgggCgCCggTgAC-3’-3’(SEQ ID NO：32)

5’-TCgTCgCCggTgggggTgTg-3’3’(SEQ ID NO：33)

5’-TCgTCgTACgCAATTgTCTT-3’3’(SEQ ID NO：34)

5’-TCgCCCACCggTgggggggg-3’3’(SEQ ID NO：35)

5’-TCgTCgCAgACCggTCTgggg-3’(SEQ ID NO：36)

5’-TCgTCgCggCCggCgCCCCC-3’(SEQ ID NO：39)

5’-TCgTCgCggCCgCgAggggg-3’(SEQ ID NO：40)

5’-TCgAggACAAgATTCTCgTgC-3’(SEQ ID NO：41)

5’-TCgAggACAAgATTCTCgTgCAggCC-3’(SEQ ID NO：42)

5’-TCgTgCAggCCAACgAggCCg-3’(SEQ ID NO：43)

5’-TCgTTgCCgTCggCCC-3’(SEQ ID NO：45)

5’-TCggCACgCgACgTgCTggCCgTCgTTTCC-3’(SEQ ID NO：47)

5’-TCgTTgCCgTCggCCCCCCCCC-3’(SEQ ID NO：54)

5’-TCgTTgCCgTCggCCCCCC-3’(SEQ ID NO：55)

5’-TCgTTgCCgTCggCCCCC-3’(SEQ ID NO：56)

5’-TCgTTgCCgTCggCCCC-3’(SEQ ID NO：57)

5’-TCgTTgCCgTCggCCCCCCC-3’(SEQ ID NO：58)

5’-TCgAggACAAgATTCTCgT-3’(SEQ ID NO：67)

5’-TCggCACgCgACgTgCTggCCgTCgTT-3’(SEQ ID NO：69)

5’-TCgTCgCgCCgTCACgggggg-3’(SEQ ID NO：70)

5’-TCgTgTgCgTgCCgTTggg-3’(SEQ ID NO：71)

5’-TCgTCgCCgTTgggCggg-3’(SEQ ID NO：72)

5’-TCgTCgACgTCgTTgggCggg-3’(SEQ ID NO：73)

5’-TCgCAgTTgTCgTAACgTTgggCggg-3’(SEQ ID NO：74)

5’-TCgTCgTTggTATgTT-3’(SEQ ID NO：77)

5’-TCgTCgTCgTCgTTgTCgTT-3’(SEQ ID NO：78)

5’-TCgTCgTCgTCgTTgTCgTTgggg-3’(SEQ ID NO：79)

5’-TCgTTCggggTgCCg-3’(SEQ ID NO：80)

5’-TCgTTCggggTAACgATT-3’(SEQ ID NO：81)

5’-TCgTTCggggTAACgTT-3’(SEQ ID NO：82)

5’-TCgTTCggggTACCgAT-3’(SEQ ID NO：83)

5’-TCgTACggCCgCCgTACggCggg-3’(SEQ ID NO：92)

5’-TCgCgTCgACTCCCCTCgAgggg-3’(SEQ ID NO：94)

5’-TCgTCgTCgACTCgTggTCggggg-3’(SEQ ID NO：95)

5’-TCgggCgCCCgATCgggggg-3’(SEQ ID NO：96)

5’-TCgTCggTCTTTCgAAATT-3’(SEQ ID NO：97)

5’-TCgTgACgTCCTCgAgTT-3’(SEQ ID NO：98)

5’-TCgTCTTTCgACTCgTTCTC-3’(SEQ ID NO：115)

5’-TCgTCgTTTTgCgTTCTC-3’(SEQ ID NO：116)

5’-TCgACTTTCgTCgTTCTgTT-3’(SEQ ID NO：119)

5’-TCgTCgTTTCgTCgTTCTC-3’(SEQ ID NO：120)

5’-TCgTCgTCgTCgTTgTCgTT-3’(SEQ ID NO：121)

5’-TCgTTCTCgACTCgTTCTC-3’(SEQ ID NO：122)

5’-TCgACgTTCgTCgTTCgTCgTTC-3’(SEQ ID NO：123)

5’-TCgTCgACgTCgTTCgTTCTC-3’(SEQ ID NO：124)

5’-TCgTgCgACgTCgCAgATgAT-3’(SEQ ID NO：138)

5’-TCgTCgAgCgCTCgATCggAT-3’(SEQ ID NO：141)

5’-TCgTCgTTTCgTAgTCgTTgACgTCggg-3’(SEQ ID NO：142)

5’-TCgTCggACgTTTTCCgACgTTCT-3’(SEQ ID NO：144)

5’-TCgTCgTTTTCgTCgTTTTCgTCgTT-3’(SEQ ID NO：151)

5’-TCgTCgTTTgTCgTgTgTCgTT-3；(SEQ ID NO：152)

5’-TCgTCgTTggTCggggTCgTTggggTCgTT-3’(SEQ ID NO：153)

5’-TCgTCgTTTCgTCTCTCgTT-3’(SEQ ID NO：154)

5’-TCgTCgTTTTgCTgCgTCgTT-3’(SEQ ID NO：155)

5’-TCgAgCgTTTTCgCTCgAATT-3’(SEQ ID NO：178)

(5) comprise the sequence of TTCGTCG

5’-TTCgTCgTTTgATCgATgTTCgTTgggggg-3’(SEQ ID NO：25)’

5’-TTCgTCgTTgTgATCgATgggggg-3’-3’(SEQ ID NO：26)

5’-TATCgATgTTTTCgTCgTCgTTgggggg-3’(SEQ ID NO：27)

5’-TCgACTTTCgTCgTTCTgTT-3’(SEQ ID NO：119)

5’-TCgTCgTTTCgTCgTTCTC-3’(SEQ ID NO：120)

5’-TCgACgTTCgTCgTTCgTCgTTC-3’(SEQ ID NO：123)

5’-TCgTCgTTTTCgTCgTTTTCgTCgTT-3’(SEQ ID NO：151)

Synthesizing of embodiment 2 CpG single chain deoxynucleotides

The synthetic advantages such as solid phase phosphoramidite triester method synthetic DNA fragment, this method has efficiently, quick coupling that adopt of DNA are extensive use of in the DNA chemosynthesis.

The DNA chemosynthesis is different from the DNA building-up process of enzymatic extends from 5 ' → 3 ' direction, but by 3 ' end beginning.Concrete reactions steps is as follows:

One, deprotection base

Slough the blocking group DMT (dimethoxytrityl) that is attached at the Nucleotide on the CpG (Controlled Pore Glass) with trichoroacetic acid(TCA), obtain free 5 '-hydroxyl terminal, for next step condensation reaction.

Two, activation

With the 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), this intermediate will with the Nucleotide generation condensation reaction of the base of deprotection on the GpG.

Three, connect

When phosphoramidite tetrazolium active intermediate runs on the CpG the Nucleotide of deprotection base, will with its 5 '-hydroxyl generation affinity reaction, condensation is also sloughed tetrazolium, this moment, the synthetic oligonucleotide chain prolonged a base forward.

Four, sealing

For 5 ' of the reaction-hydroxyl that has neither part nor lot in that prevents to be connected on the CpG is extended, often seal this terminal hydroxy group by acetylize after the condensation reaction in circulating reaction subsequently, general acetylation reagent mixes formation with diacetyl oxide and N-Methylimidazole etc.

Five, 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 instability, easily by acid, basic hydrolysis, this moment, the tetrahydrofuran solution of iodine commonly used was converted into phosphotriester with inferior phosphinylidyne, obtained stable oligonucleotide.

Through after above five steps; a deoxynucleotide is just linked on the Nucleotide of CpG; after sloughing blocking group DMT on the deoxynucleotide 5 '-hydroxyl that newly connects with trichoroacetic acid(TCA) equally again, repeat above activation, connection, sealing, oxidising process and can obtain a dna fragmentation crude product.At last to its cut, the deprotection base (generally adopts the benzoyl protection to A, C base; The G base is protected with isobutyryl; The T base needn't be protected; Phosphorous acid is with nitrile ethyl protection), purifying (commonly used have HAP, PAGE, HPLC, C18, methods such as OPC), synthetic aftertreatment such as quantitative can obtain meeting the oligonucleotide fragment of requirement of experiment.

The solid phase synthesis oligonucleotide carries out on dna synthesizer; aforesaid method synthetic oligonucleotide is after sloughing protecting group; purpose oligonucleotide purity is extremely low; contain a large amount of impurity; phenylformic acid ammonia and isopropylformic acid ammonia that protecting group that major impurity is taken off to some extent and ammonia form; nitrile ethyl of taking off on the nitrile phosphorus base and the short chain that produces when synthetic etc. are to such an extent as to oligonucleotide content only is about 15% in the thick product.Although the efficient in each step is all 97%～98% when synthetic, cumulative efficient is not high.With chain length 20mer and 50mer is example, (97.5%) 20 ≈ 60%, (97.5%) 50 ≈ 28%, as seen purpose oligonucleotide content is very low in thick product, in addition 10% less than.These impurity especially are present in a large amount of salt and short chain in the thick product, cause quantitatively and are forbidden, and influence next step reaction, therefore must carry out purifying to oligonucleotide.Polyacrylamide gel electrophoresis (PAGE) purifying is adopted in suggestion, and the product purity height of this method purifying can be used for the molecular biology experiment of exhausted major part, can avoid many beyond thought troubles.If consider reduction of expenditure,,, then adopt desalting and purifying to get final product as simple PCR reaction for requiring lower experiment.

Oligonucleotide DNA is with OD ₂₆₀Value is measured.In the 1cm of 1ml light path standard quartz cuvette, absorbancy is that 1 oligonucleotide solution is defined as 1OD under the 260nm wavelength ₂₆₀Though for every kind of specific oligonucleotide, the composition of its base is not quite similar, the weight of 1260OD oligonucleotide DNA is about 33 μ g.

Embodiment 3 contains the enhancement of the single chain deoxynucleotide of CpG to heat shock protein(HSP) C hepatitis virus antigen peptide fusion protein immunostimulation

One, hepatitis C virus specific cytotoxic T lymphocyte fragmentation test

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and multi-epitope HCV cAg fusion rotein (HSP-HCV) contain the deoxy-oligonucleotide (CpG ODN) of CpG, the B16 cell (HCV of transfection HCV cAg multi-epitope antigen peptide gene ⁺The B16 cell).CpG ODN1(SEQ ID NO：7)，CpG ODN2(SEQ ID NO：106)，CpG ODN3(SEQ ID NO：103)，CpG ODN4(SEQ ID NO：18)，CpG ODN5(SEQ ID NO：86)，CpG ODN6(SEQ ID NO：79)，CpG ODN7(SEQ ID NO：95)，CpG ODN8(SEQ ID NO：123)，CpG ODN9(SEQ ID NO：124)，CpG ODN10(SEQ ID NO：159)。

2, laboratory animal and grouping:

40 of the male C57/BL6 mouse in employing 6-8 week, every group of 10 mouse, be divided into physiological saline group (injecting normal saline), HSP-HCV organizes (injecting 20 μ g HSP-HCV), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP-HCV+CpG ODN organizes (injecting 20 μ g HSP-HCV, 50 μ g CpG ODN).HSP-HCV and CpGODN are mixed with desired concn with PBS respectively, and HSP-HCV+CpG ODN group is that HSP-HCV and CpGODN are mixed the back injection.

3, experimental technique

(1) injection

Used liquid, CpG ODN application liquid and HSP-HCV+CpG ODN mixed solution to each group injected in mice physiological saline, HSP-HCV respectively in the 1st, 14 and 21 day.

(2) preparation effector cell

At the 26th day mouse being taken off neck puts to death.Get mouse boosting cell or lymph-node cell according to a conventional method, the preparation single cell suspension.With the IMDM substratum re-suspended cell that contains 10%FBS, mouse Pi cell and conditioned medium and HSP-HCV (20 μ g/ml) that the ConA of adding 10% stimulates.37 ℃, 5%CO ₂Cultivated 7 days.The results mouse boosting cell is the effector cell.

(3) preparation target cell

Adopt the IMDM culture medium culturing HCV of 10%FBS ⁺The B16 cell.With ⁵¹Cr mark HCV ⁺The B16 cell (at 37 ℃, 5%CO ₂Cultivated 1 hour under the condition).With the IMDM washed cell that contains 10%FBS, totally three times.

(4) cellulotoxic experiment

Will ⁵¹Cr mark HCV ⁺The B16 cell joins in the hole of 96 well culture plates in the IMDM of 10%FBS substratum, and every hole 100 μ l contain 1 * 10 ⁴Individual cell.Imitate target than adding effector cell (100 μ l) by 100: 1, establish three repeating holes.37 ℃, 5%CO ₂Cultivated 4-6 hour.With centrifugal 5 minutes of 96 well culture plates (3000rpm).From every hole sucking-off 100 μ l supernatants, survey radioactivity.By following formula calculating effector cell's cytotoxic activity, represent with the specific killing rate.

The maximum spontaneous release rpm of rpm-that discharges of the spontaneous release rpm/ of specific killing rate (%)=experimental port rpm-

4, experimental result:

The results are shown in following table, the average (n=10) of 10 data represented mouse data wherein.

Table 1-1CpG ODN induces the active enhancement of specific CTL to HSP65-HCV

Group	Specific killing rate (%)

Physiological saline	0.31±0.22
		HSP-HCV	15.00±6.00

		CpG ODN1	0.51±0.33
HSP-HCV+CpG ODN1	26.00±4.12

CpGODN2	0.63±0.43
		HSP-HCV+CpG ODN2	27.32±3.23

		CpGODN3	1.02±0.56
HSP-HCV+CpG ODN3	32.22±6.78

CpG ODN4	0.88±0.54
		HSP-HCV+CpG ODN4	29.98±5.23

CpGODN5	0.76±0.77
		HSP-HCV+CpG ODN5	35.00±3.89

		CpG ODN6	0.88±0.45
HSP-HCV+CpG ODN6	34.98±5.03

CpG ODN7	1.51±1.22
		HSP-HCV+CpG ODN7	31.34±6.65

		CpG ODN8	1.25±0.89
HSP-HCV+CpG ODN8	27.75±5.65

CpG ODN9	1.02±0.67
		HSP-HCV+CpG ODN9 CpG ODN10 HSP-HCV+CpG ODN10	37.21±6.24 0.98±0.56 32.78±5.32

5, conclusion:

The single chain deoxynucleotide that contains CpG can obviously strengthen the activity (p＜0.05) that the immunity of heat shock protein(HSP) C hepatitis virus antigen peptide fusion protein induces the specific cytotoxic T lymphocyte of HCV cAg.This CTL can kill and wound the cell that HCV infects in vivo.Therefore, the single chain deoxynucleotide that contains CpG can obviously strengthen antiviral (including but not limited to hepatitis C virus) activity of heat shock protein(HSP) C hepatitis virus antigen peptide fusion protein.

Two, kill and wound the experiment of HCV cells infected in the body

B16 cell (the HCV of transfection HCV cAg multi-epitope antigen peptide gene ⁺The B16 cell) can be used as the cell model (CN02122116.2) of infection with hepatitis C virus.Observe mouse and suppress the ability of this B16 cell growth, can detect that injection contains CpG single chain deoxynucleotide (CpG ODN) and tubercule bacillus heat shock protein(HSP) 65 multi-epitope HCV cAg fusion roteins stimulation mouse CTL kills and wounds the HCV infection cell activity in vivo.

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and multi-epitope HCV cAg fusion rotein (HSP-HCV) contain the single chain deoxynucleotide (CpG ODN) of CpG, the B16 cell (HCV of transfection HCV cAg multi-epitope antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

40 of the male C57/BL6 mouse in employing 6-8 week, every group 10, be divided into physiological saline group (injecting normal saline), HSP-HCV organizes (injecting 20 μ g HSP-HCV), CpG ODN group (injecting 50 μ g CpG ODN) and HSP-HCV+CpG ODN group (are injected 20 μ g HSP-HCV, 50 μ gCpG ODN).

3, experimental technique

(1) injection

HSP-HCV and CpGODN are mixed with desired concn with PBS respectively, and HSP-HCV+CpG ODN group is that HSP-HCV and CpGODN are mixed the back injection.Organized injected in mice physiological saline, HSP-HCV, CpG ODN and HSP-HCV+CpG ODN to each respectively in the 1st, 14 and 21 day.In the 24th day inoculation HCV ⁺B16 cell (1 * 10 ⁵Individual/only), the injection site is that the right side of mice back is subcutaneous.

(2) get tumour and weighing

The weight that mouse is got tumour and takes by weighing tumour is killed in behind inoculated tumour the 15th day.

4, experimental result:

Table 1-2 CpG ODN stimulates mouse CTL to kill and wound the enhancement of HCV infection cytoactive in vivo to HSP65-HCV

Group	Tumor weight (gram)

Physiological saline	6.23±1.34
		HSP-HCV	3.02±0.63

		CpG ODN1	5.60±1.33
HSP-HCV+CpG ODN1	1.23±0.41

CpG ODN2	5.89±0.94
		HSP-HCV+CpG ODN2	1.32±0.77

		CpG ODN3	6.33±1.22
HSP-HCV+CpG ODN3	0.91±0.54

CpG ODN4	5.88±1.18
		HSP-HCV+CpG ODN4	2.01±0.12

		CpGODN5	6.17±1.34
HSP-HCV+CpG ODN5	1.35±0.34

CpGODN6	5.88±1.45
		HSP-HCV+CpG ODN6	0.77±0.22

		CpG ODN7	6.05±1.56
HSP-HCV+CpG ODN7	1.79±0.36

CpG ODN8	5.96±0.98
		HSP-HCV+CpG ODN8	0.77±0.36

		CpG ODN9	6.02±1.16
HSP-HCV+CpG ODN9 CpG ODN10 HSP-HCV+CpG ODN10	1.23±0.62 5.68±1.17 1.26±0.59

5, conclusion

The single chain deoxynucleotide (CpG ODN) that contains CpG can significantly strengthen tubercule bacillus heat shock protein(HSP) 65 multi-epitope HCV cAg fusion roteins stimulates mouse CTL to kill and wound HCV infection cell activity (p＜0.05) in vivo, and its performance is: the energy for growth that the mouse of combined utilization CpG ODN and tubercule bacillus heat shock protein(HSP) 65 multi-epitope HCV cAg fusion roteins suppresses the tumour cell of expression of HCV cAg significantly strengthens.

Embodiment 4 contains the single chain deoxynucleotide of CpG to heat shock protein(HSP) chlamydia trachomatis major outer membrane albumen epitope antigen

Peptide fusion protein induces the active enhancement of specific CTL

1, material:

Tubercule bacillus heat shock protein(HSP) 65 and chlamydia trachomatis major outer membrane proteantigen peptide fusion protein (HSP65-Chla) contain the single chain deoxynucleotide (CpG ODN) of CpG, the B16 cell (Chla of transfection chlamydia trachomatis major outer membrane proteantigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

40 of the female C57/BL6 mouse in employing 6-8 week, every group 10, be divided into physiological saline group (injecting normal saline), HSP65-Chla organizes (injecting 20 μ g HSP65-Chla), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP65-Chla+CpG ODN organizes (injecting 20 μ g HSP65-Chla, 50 μ g CpG ODN).

3, experimental technique

(1) injection

HSP65-Chla and CpGODN are mixed with desired concn with PBS respectively, and HSP65-Chla+CpG ODN group is that HSP65-Chla and CpGODN are mixed the back injection.Organized injected in mice physiological saline, HSP65-Chla, CpG ODN and HSP65-Chla+CpG ODN to each respectively in the 1st, 14 and 21 day.

(2) preparation effector cell

At the 26th day mouse being taken off neck puts to death.Get mouse boosting cell or lymph-node cell according to a conventional method, below operation is as the relevant statement of embodiment 1.

(3) preparation target cell

Adopt the IMDM culture medium culturing Chla of 10%FBS ⁺The B16 cell.With ⁵¹The Chla of Cr mark ⁺The B16 cell is done target cell, the relevant statement of following operation such as embodiment 1.

(4) cellulotoxic experiment

Will ⁵¹The Chla of Cr mark ⁺The B16 cell joins in the hole of 96 well culture plates in the IMDM of 10%FBS substratum, the relevant statement of following operation and cytotoxic calculating such as embodiment 1.

4, experimental result:

Table 2CpG ODN induces the active enhancement of specific CTL to HSP-Chla

Group	Specific killing rate (%)

Physiological saline	1.49±0.92
		HSP-Chla	10.13±4.49

		CpG ODN1	2.53±1.22
HSP-Chla+CpG ODN1	16.47±3.91


		CpG ODN2	0.79±0.62
HSP-Chla+CpG ODN2	17.68±5.12

CpG ODN3	1.02±0.67
		HSP-Chla+CpG ODN3	27.28±5.19

		CpG ODN4	1.80±0.57
HSP-Chla+CpG ODN4	25.59±3.95

CpG ODN5	0.94±0.87
		HSP-Chla+CpG ODN5	31.34±6.18

		CpG ODN6	1.23±1.11
HSP-Chla+CpG ODN6	23.27±4.16

CpG ODN7	1.51±0.87
		HSP-Chla+CpG ODN7	28.34±6.31

		CpG ODN8	0.79±0.45
HSP-Chla+CpG ODN8	27.95±5.93

CpG ODN9	1.76±1.01
		HSP-Chla+CpG ODN9 CpG ODN 10 HSP-Chla+CpG ODN 10	30.07±4.99 1.48±0.89 29.98±5.76

5, conclusion

The single chain deoxynucleotide that contains CpG can obviously strengthen the activity (p＜0.05) that tubercule bacillus heat shock protein(HSP) 65 and chlamydia trachomatis major outer membrane proteantigen peptide fusion protein induce the chlamydozoan specific CTL.This CTL can kill and wound in vivo and infect chlamydial cell.Therefore, the single chain deoxynucleotide that contains CpG can obviously strengthen the activity of tubercule bacillus heat shock protein(HSP) 65 and the treatment of chlamydia trachomatis major outer membrane proteantigen peptide fusion protein and prevention choamydiae infection and relative disease thereof.

Embodiment 5 contains the single chain deoxynucleotide of CpG to tubercule bacillus heat shock protein(HSP) 65 and human prostate-specific antigen (Prostate specific antigen, PSA) enhancement of epitope antigen peptide fusion protein immunostimulation

One, CpG ODN induces the active enhancement of specific CTL to HSP65-HCV

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and human prostate-specific antigen (Prostate specificantigen, PSA) antigen peptide fusion rotein (HSP-PSA), the single chain deoxynucleotide (CpG ODN) that contains CpG, the B16 cell (PSA of transfection PSA antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

40 of the male C57/BL6 mouse in employing 6-8 week, every group 10, be divided into physiological saline group (injecting normal saline), HSP-PSA organizes (injecting 20 μ g HSP-PSA), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP-PSA+CpG ODN organizes (injecting 20 μ g HSP-PSA, 50 μ g CpG ODN).

3, experimental technique

(1) injection

Organized injected in mice physiological saline, HSP-PSA, CpG ODN and HSP-PSA+CpG ODN to each respectively in the 1st, 14 and 21 day.

(2) preparation effector cell

At the 26th day mouse being taken off neck puts to death.Get mouse boosting cell or lymph-node cell according to a conventional method, the preparation single cell suspension.With the IMDM substratum re-suspended cell that contains 10%FBS, mouse Pi cell conditioned medium and HSP-PSA (200 μ g/ml) that the ConA of adding 10% stimulates.37 ℃, 5%CO ₂Cultivated 7 days.Harvested cell is the effector cell.

(3) preparation target cell

Employing contains the B16 cell (PSA of the IMDM culture medium culturing transfection PSA antigen peptide gene of 10%FBS ⁺The B16 cell).With ⁵¹Cr mark PSA ⁺The B16 cell, (cultivated 1 hour, 37 ℃, 5%CO ₂).With the IMDM washed cell that contains 10%FBS, totally three times.

(4) cellulotoxic experiment

Will ⁵¹Cr mark PSA ⁺The B16 cell joins in the hole of 96 well culture plates in the IMDM of 10%FBS substratum, and every hole 100 μ l contain 1 * 10 ⁴Individual cell.Imitate target than adding the effector cell by 100: 1, establish three multiple holes.The relevant statement of following operation and cytotoxic calculating such as embodiment 1.

4, experimental result:

Table 3-1CpG ODN induces the active enhancement of specific CTL to HSP-PSA

Group	Specific killing rate (%)

Physiological saline	0.22±0.19
		HSP-PSA	12.39±4.67


		CpG ODN1	0.27±0.23
HSP-PSA+CpG ODN1	21.39±5.45

CpG ODN2	0.55±0.37
		HSP-PSA+CpG ODN2	22.89±4.12


		CpG ODN3	0.70±0.41
HSP-PSA+CpG ODN3	19.85±5.27

CpGODN4	0.83±0.65
		HSP-PSA+CpG ODN4	32.32±5.69

		CpG ODN5	0.45±0.31
HSP-PSA+CpG ODN5	23.00±2.98

CpG ODN6	1.04±0.34
		HSP-PSA+CpG ODN6	31.22±5.12

		CpG ODN7	1.51±1.22
HSP-PSA+CpG ODN7	31.34±6.65

CpG ODN8	0.89±0.65
		HSP-PSA+CpG ODN8	24.57±4.89

		CpG ODN9	0.74±0.61
HSP-PSA+CpG ODN9 CpG ODN10 HSP-PSA+CpG ODN10	27.02±5.51 0.84±0.58 29.02±4.62

5, conclusion:

The single chain deoxynucleotide that contains CpG can obviously strengthen the activity (p＜0.05) that tubercule bacillus heat shock protein(HSP) 65 and PSA antigen peptide fusion protein immunization induce the PSA specific CTL, and this CTL can kill and wound the tumour cell of expressing PSA.Therefore, the single chain deoxynucleotide that contains CpG can obviously strengthen the biologic activity of prevention of heat shock protein(HSP) PSA antigen peptide fusion rotein and treatment prostate cancer.

Two, CpG ODN is to the anti-enhancement of expressing the PSA tumor promotion of HSP65-PSA

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and human prostate-specific antigen (Prostate specific antigen, PSA) antigen peptide fusion rotein (HSP-PSA), the single chain deoxynucleotide (CpG ODN) that contains CpG, the B16 cell (PSA of transfection PSA antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

3, experimental technique

(1) injection

HSP-PSA and CpG ODN prepare with PBS, and HSP-PSA+CpG ODN group is that HSP-PSA and CpG ODN are mixed the back injection.Gave injected in mice physiological saline, HSP-PSA, CpG ODN and HSP-PSA+CpG ODN respectively in the 1st, 14 and 21 day.In the 28th day inoculation PSA ⁺B16 cell (1 * 10 ⁵/ only), the injection site is that the right back back of mouse is subcutaneous.

(2) get tumour and weighing

The weight that mouse is got tumour and claims tumour is killed in behind inoculated tumour the 15th day.

4, experimental result:

Table 3-2CpG ODN is to the anti-enhancement of expressing the PSA tumor promotion of HSP65-PSA

Group	Tumor weight (gram)

Physiological saline	5.12±1.22
		HSP-PSA	3.33±0.61

		CpG ODN1	4.60±1.00
HSP-PSA+CpG ODN1	1.12±0.33

CpG ODN2	5.00±0.99
		HSP-PSA+CpG ODN2	1.11±0.65

		CpG ODN3	4.91±1.00
HSP-PSA+CpG ODN3	0.98±0.48

CpG ODN4	4.88±0.87
		HSP-PSA+CpG ODN4	1.01±0.34

		CpG ODN5	5.17±0.73

HSP-PSA+CpG ODN5	0.98±0.51

CpG ODN6	4.77±0.98
		HSP-PSA+CpG ODN6	1.30±0.33

		CpG ODN7	5.05±1.21
HSP-PSA+CpG ODN7	1.66±0.41

CpG ODN8	4.99±1.01
		HSP-PSA+CpG ODN8	0.88±0.41

		CpG ODN9	4.88±1.02
HSP-PSA+CpG ODN9 CpG ODN10 HSP-PSA+CpG ODN 10	1.12±0.60 5.38±1.22 1.34±0.58

5, conclusion

The single chain deoxynucleotide that contains CpG can obviously strengthen tubercule bacillus heat shock protein(HSP) 65 and suppress to express the growth (p＜0.05) of PSA tumour cell with PSA antigen peptide fusion rotein.The single chain deoxynucleotide that contains CpG can obviously strengthen the biologic activity of heat shock protein(HSP) and prevention of PSA antigen peptide fusion rotein and treatment prostate cancer.

Embodiment 6 contains the enhancement of the single chain deoxynucleotide of CpG to heat shock protein(HSP)-MUC1 antigen peptide fusion protein immunization hormesis

One, CpG ODN induces the active enhancement of specific CTL to HSP65-MUC1

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and MUC1 antigen peptide fusion rotein (HSP-MUC1) contain the single chain deoxynucleotide (CpG ODN) of CpG, the B16 cell (MUC1 of transfection MUC1 antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

40 of the female C57/BL6 mouse in employing 6-8 week, every group 10, be divided into physiological saline group (injecting normal saline), HSP-MUC1 organizes (injecting 20 μ g HSP-MUC1), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP-MUC1+CpGODN organizes (injecting 20 μ g HSP-MUC1,50 μ g CpG ODN).

3, experimental technique

(1) injection

HSP-MUC1 and CpG ODN prepare to desired concn with PBS, and HSP-MUC1+CpG ODN group is that HSP-MUC1 and CpG ODN are mixed the back injection.Organized injected in mice physiological saline, HSP-MUC1, CpG ODN and HSP-MUC1+CpG ODN to each respectively in the 1st, 14 and 21 day.

(2) preparation effector cell

At the 26th day mouse being taken off neck puts to death.Get mouse boosting cell or lymph-node cell according to a conventional method, the preparation single cell suspension.With the IMDM substratum re-suspended cell that contains 10%FBS, mouse Pi cell conditioned medium and HSP-MUC1 (200 μ g/ml) that the ConA of adding 10% stimulates.37 ℃, 5%CO ₂Cultivated 7 days.Harvested cell is the effector cell.

(3) preparation target cell

Adopt the IMDM culture medium culturing MUC1 of 10%FBS ⁺The B16 cell.With ⁵¹Cr mark PSA ⁺The B16 cell (cultivated 1 hour, 37 ℃, 5%CO ₂).With the IMDM washed cell that contains 10%FBS, totally three times.

(4) cellulotoxic experiment

Will ⁵¹Cr mark MUC1 ⁺The B16 cell joins in the hole of 96 well culture plates in the IMDM of 10%FBS substratum, and every hole 100 μ l contain 1 * 10 ⁴Individual cell.Imitate target than adding the effector cell by 100: 1, establish three multiple holes.The relevant statement of following operation and cytotoxic calculating such as embodiment 1.

4, experimental result:

Table 4-1CpG ODN induces the active enhancement of specific CTL to HSP65-MUC1

Group	Specific killing rate (%)

Physiological saline	0.46±0.43
		HSP-MUC1	14.23±3.56

		CpG ODN1	0.59±0.44
HSP-MUC1+CpG ODN1	20.43±4.91

CpG ODN2	0.41±0.31
		HSP-MUC1+CpG ODN2	25.12±5.13

		CpGODN3	0.65±0.32
HSP-MUC1+CpG ODN3	21.23±4.91

CpG ODN4	0.77±0.49
		HSP-MUC1+CpG ODN4	25.01±4.15

		CpG ODN5	0.51±0.42
HSP-MUC1+CpG ODN5	29.12±3.19

CpG ODN6	0.74±0.52

HSP-MUC1+CpG ODN6

28.82±4.09


		CpG ODN7	1.01±0.78
HSP-MUC1+CpG ODN7	27.65±4.97

CpG ODN8	0.76±0.47
		HSP-MUC1+CpG ODN8	29.11±5.17

		CpG ODN9	0.65±0.34
HSP-MUC1+CpG ODN9 CpG ODN10 HSP-MUC1+CpG ODN10	22.23±4.15 0.85±0.44 26.22±5.13

5, conclusion:

The single chain deoxynucleotide that contains CpG can obviously strengthen the activity (p＜0.05) that tubercule bacillus heat shock protein(HSP) 65-MUC1 antigen peptide fusion rotein induces the MUC1 specificity cell toxicity T lymphocyte, and this CTL can kill and wound the tumour cell of expressing MUC1.Therefore, the single chain deoxynucleotide that contains CpG can obviously strengthen the biologic activity that MUC1 tumour such as mammary cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer etc. are expressed in the prevention of tubercule bacillus heat shock protein(HSP) 65-MUC1 antigen peptide fusion rotein and treatment.

Two, CpG ODN is to the anti-enhancement of expressing the MUC1 tumor promotion of HSP65-MUC1

1, material:

2, laboratory animal and grouping: the female C57/BL6 mouse of adopting 6-8 week, be divided into physiological saline group (injecting normal saline), HSP-MUC1 organizes (injecting 20 μ g HSP-MUC1), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP-MUC1+CpG ODN group (is injected 20 μ g HSP-MUC1,50 μ g CpG ODN), 10 every group.

3, experimental technique

(1) injection

In the 28th day inoculation MUC1 ⁺B16 cell (1 * 10 ⁵/ only), the injection site is that the right back back of mouse is subcutaneous.

(2) get tumour and weighing

Behind inoculated tumour the 15th day killed mouse and claimed the weight of tumour.

4, experimental result:

Table 4-2CpG ODN is to the anti-enhancement of expressing the MUC1 tumor promotion of HSP65-MUC1

Group	Tumor weight (gram)

Physiological saline	5.68±1.34
		HSP-MUC1	3.57±0.57

		CpG ODN1	5.60±1.12
HSP-MUC1+CpG ODN1	2.01±0.45

CpG ODN2	5.00±0.99
		HSP-MUC1+CpG ODN2	1.11±0.65

		CpG ODN3	4.99±1.56
HSP-MUC1+CpG ODN3	1.23±0.38

CpG ODN4	5.55±0.89

HSP-MUC1+CpG ODN4	1.13±0.23

CpG ODN5	5.45±1.66
		HSP-MUC1+CpG ODN5	1.39±0.50

		CpG ODN6	5.37±1.65
HSP-MUC1+CpG ODN6	1.32±0.41

CpG ODN7	5.33±1.43
		HSP-MUC1+CpG ODN7	1.35±0.36

		CpG ODN8	5.29±0.89
HSP-MUC1+CpG ODN8	1.31±0.50

CpG ODN9	4.99±1.56
		HSP-MUC1+CpG ODN9 CpG ODN10 HSP-MUC1+CpG ODN10	1.09±0.55 5.32±1.43 1.12±0.47

5, conclusion

The single chain deoxynucleotide that contains CpG can obviously strengthen the growth (p＜0.05) that tubercule bacillus heat shock protein(HSP) 65-MUC1 antigen peptide fusion rotein suppresses to express the MUC1 tumour cell.The single chain deoxynucleotide that contains CpG can obviously strengthen the biologic activity that MUC1 tumour such as mammary cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer etc. are expressed in the prevention of tubercule bacillus heat shock protein(HSP) 65-MUC1 antigen peptide fusion rotein and treatment.

Embodiment 7 contains the enhancement of the single chain deoxynucleotide of CpG to heat shock protein(HSP) HER2 antigen peptide fusion protein immunization hormesis

One, CpG ODN induces the active enhancement of specific CTL to HSP65-HER-2

1, material:

Tubercule bacillus heat shock protein(HSP) 65 and HER-2 antigen peptide fusion rotein (HSP-HER-2) contain the single chain deoxynucleotide (CpG ODN) of CpG, the B16 cell (HER-2 of transfection HER-2 antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

Adopt the female C57/BL6 mouse in 6-8 week, be divided into physiological saline group (injecting normal saline), HSP-HER-2 organizes (injecting 20 μ g HSP-HER-2), CpG ODN organizes (injecting 50 μ g CpG ODN), HSP-HER-2+CpG ODN group (is injected 20 μ g HSP-HER-2,50 μ g CpG ODN), 10 every group.

3, experimental technique

(1) injection

HSP-HER-2 and CpG ODN prepare with PBS, and HSP-HER-2+CpG ODN mixes the back injection with HSP-HER-2 and CpG ODN.Gave injected in mice physiological saline, HSP-HER-2, CpG ODN and HSP-HER-2+CpG ODN respectively in the 1st, 14 and 21 day.

(2) preparation effector cell

At the 26th day mouse being taken off neck puts to death.Get mouse boosting cell or lymph-node cell according to a conventional method, the preparation single cell suspension.With the IMDM substratum re-suspended cell that contains 10%FBS, mouse Pi cell conditioned medium and HSP-HER-2 (200 μ g/ml) that the ConA of adding 10% stimulates.37 ℃, 5%CO ₂Cultivated 7 days.Harvested cell is the effector cell.

(3) preparation target cell

Adopt the B16 cell (HER-2 of the IMDM culture medium culturing transfection HER-2 antigen peptide gene of 10%FBS ⁺The B16 cell).With ⁵¹Cr mark HER-2 ⁺The B16 cell (cultivated 1 hour, 37 ℃, 5%CO ₂).With the IMDM washed cell that contains 10%FBS, totally three times.

(4) cellulotoxic experiment

Will ⁵¹Cr mark HER-2 ⁺The B16 cell joins in the hole of 96 well culture plates in the IMDM of 10%FBS substratum, and every hole 100 μ l contain 1 * 10 ⁴Individual cell.Imitate target than adding the effector cell by 100: 1, establish three multiple holes.

The relevant statement of following operation and cytotoxic calculating such as embodiment 1.

4, experimental result:

Table 5-1CpG ODN induces the active enhancement of specific CTL to HSP65-HHER-2

Group	Specific killing rate (%)

Physiological saline	0.39±0.37
		HSP-HER-2	16.34±3.39

		CpG ODN1	0.46±0.33
HSP-HER-2+CpG ODN1	19.95±3.89

CpG ODN2	0.53±0.29
		HSP-HER-2+CpG ODN2	22.33±4.97

		CpGODN3	0.71±0.29
HSP-HER-2+CpG ODN3	31.19±5.19

CpG ODN4	0.63±0.42
		HSP-HER-2+CpG ODN4	22.23±3.91

		CpG ODN5	0.45±0.37
HSP-HER-2+CpG ODN5	24.54±3.94

CpG ODN6	0.57±0.49
		HSP-HER-2+CpG ODN6	25.79±3.39

		CpG ODN7	0.98±0.55
HSP-HER-2+CpG ODN7	29.22±4.12

CpG ODN8	0.66±0.50
		HSP-HER-2+CpG ODN8	23.32±5.01

		CpG ODN9	0.61±0.39
HSP-HER-2+CpG ODN9 CpG ODN10 HSP-HER-2+CpG ODN 10	24.19±3.92 0.78±0.37 23.87±3.78

5, conclusion:

The single chain deoxynucleotide that contains CpG can obviously strengthen the activity (p＜0.05) that tubercule bacillus heat shock protein(HSP) and HER-2 antigen peptide fusion protein immunization induce the HER-2 specificity cell toxicity T lymphocyte, and this CTL can kill and wound the tumour cell of expressing HER-2.Therefore, the single chain deoxynucleotide that contains CpG can obviously strengthen the biologic activity that HER-2 tumour such as mammary cancer and ovarian cancer etc. are expressed in tubercule bacillus heat shock protein(HSP) 65 and prevention of HER-2 antigen peptide fusion rotein and treatment.

Two, CpG ODN is to the anti-enhancement of expressing the HER-2 tumor promotion of HSP65-HER-2

1, material:

Tubercule bacillus heat shock protein(HSP) 65 (HSP65) and HER-2 antigen peptide fusion rotein (HSP-HER-2) contain the single chain deoxynucleotide (CpG ODN) of CpG, the B16 cell (HER-2 of transfection HER-2 antigen peptide gene ⁺The B16 cell).

2, laboratory animal and grouping:

3, experimental technique

(1) injection

Gave injected in mice physiological saline, HSP-HER-2, CpG ODN and HSP-HER-2+CpG ODN respectively in the 1st, 14 and 21 day.In the 28th day inoculation HER-2 ⁺B16 cell (1 * 10 ⁵/ only), the injection site is that the right back back of mouse is subcutaneous.

(2) get tumour and weighing

4, experimental result:

Table 5-2CpG ODN is to the anti-enhancement of expressing the HER-2 tumor promotion of HSP65-HER-2

Group	Tumor weight (gram)

Physiological saline	4.96±1.22
		HSP-HER2	3.22±0.51

		CpG ODN1	5.00±1.01
HSP-HER2+CpG ODN 1	1.47±0.34

CpG ODN2	4.87±1.11
		HSP-HER2+CpG ODN2	0.89±0.54

CpG ODN3	4.55±1.03
		HSP-HER2+CpG ODN3	0.87±0.35

		CpG ODN4	4.80±0.76
HSP-HER2+CpG ODN4	0.56±0.52

CpG ODN5	5.04±1.26
		HSP-HER2+CpG ODN5	1.27±0.49

		CpGODN6	3.98±1.43
HSP-HER2+CpG ODN6	0.79±0.35

CpG ODN7	5.13±0.89

HSP-HER2+CpG ODN7	1.13±0.22

CpG ODN8	4.76±1.16
		HSP-HER2+CpG ODN8	0.95±0.29

		CpG ODN9	4.75±1.25
HSP-HER2+CpG ODN9 CpG ODN10 HSP-HER2+CpG ODN10	0.79±0.68 5.12±1.34 0.86±0.48

5, conclusion:

The single chain deoxynucleotide that contains CpG can obviously strengthen tubercule bacillus heat shock protein(HSP) 65 and suppress to express the growth (p＜0.05) of HER-2 tumour cell with HER-2 antigen peptide fusion rotein.The single chain deoxynucleotide that contains CpG can obviously strengthen tubercule bacillus heat shock protein(HSP) 65 and prevent and treat the biologic activity of expressing HER-2 tumour such as mammary cancer and ovarian cancer etc. with HER-2 antigen peptide fusion rotein.

Sequence table

Figure DEST_PATH_G05102957020050525D000011

Figure DEST_PATH_G05102957020050525D000021

Figure DEST_PATH_G05102957020050525D000031

Figure DEST_PATH_G05102957020050525D000051

Figure DEST_PATH_G05102957020050525D000091

Figure DEST_PATH_G05102957020050525D000111

Figure DEST_PATH_G05102957020050525D000131

Figure DEST_PATH_G05102957020050525D000151

Figure DEST_PATH_G05102957020050525D000161

Figure DEST_PATH_G05102957020050525D000171

Claims

One kind can enhancement antigen or the single chain deoxynucleotide that contains CpG of antigen composition immunostimulation, it is characterized in that described single chain deoxynucleotide is selected from the sequence of SEQ ID NO:123,124 shown in arbitrary.
2. single chain deoxynucleotide according to claim 1, described single chain deoxynucleotide also is included in the modification of each group on its base.
3. single chain deoxynucleotide according to claim 2, described modification comprise that non-thio-modification, thio-modification, part thio-modification, rare base are modified, modification methylates.
4. according to arbitrary described single chain deoxynucleotide in the claim 1 to 3, described antigen or antigen composition are that heat shock protein(HSP) and antigen peptide merge formed fusion rotein.
5. single chain deoxynucleotide according to claim 4, described heat shock protein(HSP) are tubercule bacillus heat shock protein(HSP) 65.
6. single chain deoxynucleotide according to claim 4, described antigen peptide are selected from multi-epitope core antigen of C type hepatitis virus peptide, chlamydia trachomatis major outer membrane albumen epitope antigen peptide, human prostate-specific antigen peptide, MUC1 antigen peptide or HER-2 antigen peptide.
7. a vaccine composition comprises the arbitrary described single chain deoxynucleotide of claim 1 to 3 and antigen or antigen composition.
8. vaccine composition according to claim 7, described antigen or antigen composition are that heat shock protein(HSP) and antigen peptide merge formed fusion rotein.
9. vaccine composition according to claim 8, described heat shock protein(HSP) are tubercule bacillus heat shock protein(HSP) 65.
10. vaccine composition according to claim 8, described antigen peptide are multi-epitope core antigen of C type hepatitis virus peptide.
11. vaccine composition according to claim 8, described antigen peptide are chlamydia trachomatis major outer membrane albumen epitope antigen peptide.
12. vaccine composition according to claim 8, described antigen peptide behaviour prostate specific antigen peptide.
13. vaccine composition according to claim 8, described antigen peptide are the MUC1 antigen peptide.
14. vaccine composition according to claim 8, described antigen peptide are the HER-2 antigen peptide.
15. a method of producing the described vaccine composition of claim 7 comprises the arbitrary described single chain deoxynucleotide of claim 1 to 3 is mixed with antigen or antigen composition.
16. being heat shock protein(HSP) and antigen peptide, method according to claim 15, described antigen or antigen composition merge formed fusion rotein.
17. method according to claim 16, described heat shock protein(HSP) are tubercule bacillus heat shock protein(HSP) 65.
18. method according to claim 16, described antigen peptide are multi-epitope core antigen of C type hepatitis virus peptide.
19. method according to claim 16, described antigen peptide are chlamydia trachomatis major outer membrane albumen epitope antigen peptide.
20. method according to claim 16, described antigen peptide behaviour prostate specific antigen peptide.
21. method according to claim 16, described antigen peptide are the MUC1 antigen peptide.
22. method according to claim 16, described antigen peptide are the HER-2 antigen peptide.
23. arbitrary described vaccine composition is used for the treatment of application in the vaccine of virus infection, infectation of bacteria, parasitic infection, transformation reactions or cancer in preparation in the claim 7 to 14.
24. according to the application of claim 23, described virus infection is infection with hepatitis C virus or the relative disease that caused by this virus infection.
25. according to the application of claim 23, described infectation of bacteria is choamydiae infection or the relative disease that caused by choamydiae infection.
26. according to the application of claim 23, described cancer is prostate cancer, mammary cancer, ovarian cancer, lung cancer, cancer of the stomach, carcinoma of endometrium, salivary-gland carcinoma, gland cancer, colon and the rectum cancer, nonsmall-cell lung cancer or adenocarcinoma of lung.