WO2006079291A1 - Artificial synthetic single strand deoxynucleotide and vaccine composition thereof and the use - Google Patents

Artificial synthetic single strand deoxynucleotide and vaccine composition thereof and the use Download PDF

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WO2006079291A1
WO2006079291A1 PCT/CN2006/000183 CN2006000183W WO2006079291A1 WO 2006079291 A1 WO2006079291 A1 WO 2006079291A1 CN 2006000183 W CN2006000183 W CN 2006000183W WO 2006079291 A1 WO2006079291 A1 WO 2006079291A1
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antigen
seq
cancer
peptide
heat shock
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PCT/CN2006/000183
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Chinese (zh)
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Li-Ying Wang
Mu-Sheng Bao
Yong-Li Yu
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Changchun Huapu Biotechnology Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids

Definitions

  • the present invention relates to a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory action of an antigen or antigen composition, and a vaccine composition comprising the single-stranded deoxynucleotide and an antigen or antigen composition. Furthermore, the invention relates to the use of synthetic single-stranded deoxynucleotides for the preparation of vaccine compositions, and to the use of said vaccine compositions as medicaments. Background technique
  • CpG is a dinucleotide in which cytosine and guanine are linked by a phosphate, wherein C represents cytosine, G represents guanine, p represents phosphoric acid, and cytosine is located at the 5' end.
  • CpG 0DN artificially synthesized single-stranded DNA
  • CpG 0DN single-stranded DNA
  • enhancing B cells, T cells, NK cells, and antigen presenting cells Such as monocytes, macrophages and dendritic cells
  • neutrophil activity and showed significant clinical application prospects (Weiner GJ, The iramunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol 2000 Oct; 68 (4) : 455-63).
  • synthetic single-stranded oligonucleotides can take a wide variety of forms due to the different sequences.
  • Heat shock protein is a family of molecular companion proteins found in many organisms. In the process of immune response, heat shock proteins can exhibit the following four basic functions: 1. Carrying antigens into antigen-presenting cells including dendritic cells; 2. Directing antigens into antigen-presenting cells into MHC Class I pathway processing and presentation; 3. Stimulation of antigen-presenting cells including dendritic cells to express costimulatory molecules, secreting cytokines; 4. Antigens that bind antigens and heat shock proteins to form complexes or fusion proteins, Obtaining the property of activating antigen-specific cytotoxic T lymphocytes (Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997 Oct 3 ; 278 (5335) : 117-20 ).
  • Cytotoxic T lymphocyte is one of the most powerful individuals to kill tumor cells and kill cells infected with virus-infected cells.
  • Exogenous protein antigens are mainly processed by MHC class II pathway in antigen-presenting cells including dendritic cells after application of the individual, and stimulate humoral immune response (Heikema A, Agsteribbe E, Wilschut J, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1 ; 57 (1-3) : 69-74), can not effectively induce antigen-specific CTL o
  • Mycobacterial heat shock protein 65 is capable of carrying an antigenic peptide fused or conjugated or coupled thereto into the MHC class I antigen-presenting pathway, activating antigen-peptide-specific cytotoxic T lymphocytes, and killing tumor cells expressing the antigen peptide.
  • Hepatitis C virus is the causative agent of hepatitis C and has infected more than 170 million people worldwide. Liver cancer and cirrhosis are two serious complications caused by HCV infection. Worldwide, the combination of alpha-interferon and ribavirin is a method of treating hepatitis C virus infection with an effective rate of approximately 40% (Jon Cohen. The Scientific Challenge of Hepatitis C. Science, 1999, 285: 26) - 30).
  • the fusion protein formed by the fusion of Mycobacterium tuberculosis heat shock protein 65 (HSP65) and the multi-epitope HCV core antigen is a genetically engineered recombinant protein vaccine for preventing and treating hepatitis C (CN02122116. 2, China). '
  • Chlamydia Trachomatis is a non-sports bacterium that specializes in intracellular parasitic microorganisms, which can cause trachoma, inclusion body conjunctivitis, genitourinary diseases, sexually transmitted diseases, lymphogranuloma, endometritis, salpingitis, pelvic cavity in humans. Inflammation, tubal infertility and ectopic pregnancy of the fallopian tube.
  • the major outer membrane protein (M0MP) of Chlamydia trachomatis stimulates protective immunity.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein epitope antigen peptide is a recombinant protein which has preventive and therapeutic effects on human Chlamydia trachomatis infection and related diseases (CN02141977. 9) , China).
  • Human Prostate specific antigen is a tissue-specific antigen expressed in prostate cancer cells.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and PSA epitope antigen peptide is a recombinant protein for preventing and treating human prostate cancer (CN01134935. 2, China).
  • the MUC1 protein is a type of mucins that are expressed at high levels in breast cancer, ovarian cancer, lung cancer, prostate cancer, and colorectal cancer cells, and are targets for immunotherapy.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and MUC1 antigen peptide is a tumor that expresses MUC1 protein (including but not limited to breast) Cancer) Recombinant protein with preventive and therapeutic effects (CN01.102614 6, China).
  • HER - 2 protein (HER - 2) is an epidermal growth factor receptor (Tadashi Yamamoto, et al. Similarity of protein encoded by the human c - erbB-2 gene to epidermal growth factor receptor. Nature vol 319 16 January 1986 , 230-234) Transmembrane proteins with high homology, overexpressed in 30% of human breast tumor cells, are targets for breast cancer immunotherapy.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide is a recombinant protein that has preventive and therapeutic effects on human breast cancer (CN01136347. 9, China).
  • One of the objects of the present invention is to provide a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory effect of an antigen or antigen composition, which can be used as an adjuvant for an antigen or antigen composition to enhance an antigen or antigen composition.
  • Immune stimulation consist of single-stranded DNA molecules containing one or more synthetic oligonucleotides, which may be partially sulfurized, fully vulcanized, or unvulcanized.
  • the artificially synthesized single-stranded deoxynucleotide of the present invention comprises at least one sequence selected from any one of the following formulas (1) to (5):
  • the artificially synthesized single-stranded deoxynucleotide of formula (2) has a sequence selected from any one of the following: 5, -ggggACgATACgTCggggggg-3 ' (SEQ ID N0: 1)
  • Ggggggggggggg)g (Qg : IDO1073SE NATCACACATTT- --- ggggggggggg)g (Q63 NO :10TSE IDTTCcccAA- -- 5, -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 143)
  • the synthetic single-stranded deoxynucleotide has a sequence selected from any one of the following:
  • the artificially synthesized single-stranded deoxynucleotide of formula (4) has a sequence selected from any one of the following -
  • the artificially synthesized single-stranded deoxynucleotide of formula (5) has a sequence selected from any one of the following: 5' - TTCgTCgTTTgATCgATgTTCgTTgggggg- 3, (SEQ ID NO: 25) '
  • the artificially synthesized single-stranded deoxynucleotide of the present invention further comprises a modification of each group in the base of the single-stranded deoxynucleotide, wherein the modification includes a non-thio modification, a thio modification, a partial thio Modifications, rare base modifications (dl, dU, etc.), methylation modifications, thiol groups, Aminol inker C6s or Thiol-C6 S-S, etc., are used in conjunction with other materials. Such modifications are well known to those of ordinary skill in the art.
  • Another object of the present invention is to provide a vaccine composition
  • a vaccine composition comprising the above-described single-stranded deoxynucleotide and an antigen or antigen composition, wherein the antigen or antigen composition is preferably formed of mycobacterial heat shock protein 65 and an antigen peptide A complex or fusion protein that is a preparation that induces antigen-specific CTL.
  • the antigenic peptide refers to a peptide segment having immunogenicity or a peptide having immunogenicity in a state of forming a complex, a conjugate or a fusion protein with other proteins, for example: a multi-table in 122116.
  • Hepatitis C virus core antigen antigen peptide which has preventive and therapeutic effects on diseases caused by hepatitis C virus infection and hepatitis C virus infection, and the specific sequence is as follows:
  • Glu lie Asp Asn Glu (SEQ ID NO: 181 )
  • the Chlamydia trachomatis in 9 is mainly a multi-epitope antigen polypeptide, which has therapeutic and preventive effects on related diseases caused by infection of Chlamydia and Chlamydia, and the specific sequences are as follows: Glu Phe Pro Ala Tyr Gly Arg His Met Gin Asp Ala Glu Met Phe Thr Asn Ala Ala Cys Met Ala Leu Asn lie Trp Asp Glu Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr lie 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 lie Asp Tyr His Glu Trp Gin Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr lie Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp Thr Tyr Lys Leu (SEQ ID NO : 182)
  • Human prostate specific antigen cytotoxicity T lymphocyte multi-epitope single copy antigen polypeptide which has preventive and therapeutic effects on human prostate cancer, and the specific sequence is as follows:
  • MUC1 antigen cytotoxic T lymphocyte epitope antigen peptide in CN011026146 which has preventive and therapeutic effects on tumors expressing MUC1 including, but not limited to, breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer cells, etc., specific sequence as follows:
  • Another object of the present invention is to provide a method of producing a vaccine composition comprising mixing an effective amount of the above-described synthetic single-stranded deoxynucleotide with an effective amount of an antigen or antigen composition.
  • an effective amount of the synthetic single-stranded deoxynucleotide is mixed with an effective amount of a complex or fusion protein formed by the mycobacterial heat shock protein 65 and the antigenic peptide.
  • Another aspect of the invention provides a method of enhancing the immunostimulatory effect of an antigen or antigen composition by using a synthetic single-stranded deoxynucleotide in combination with an antigen or antigen composition.
  • the synthetic single-stranded deoxynucleotide can significantly enhance the activity of the mycobacterial heat shock protein 65 antigen peptide fusion protein to induce antigenic peptide-specific CTL, which is significantly enhanced.
  • Mycobacterial heat shock protein 65 antigen peptide fusion protein stimulates mouse to inhibit the growth of antigenic peptide-expressing tumor cells, and significantly enhances the anti-tumor, anti-virus and anti-tumor specificity of mycobacterial heat shock protein 65 antigen peptide fusion protein-induced antigen peptide Anti-Chlamydia infection activity.
  • a further aspect of the invention is the use of a vaccine composition for the preparation of a vaccine for the treatment of a viral infection, a bacterial infection, a parasitic infection, an allergy or cancer.
  • This includes administering an effective amount of an immunological composition consisting of a synthetic single-stranded deoxynucleotide to an antigen or antigen composition to a human or mammal in need of treatment.
  • the viral infection, bacterial infection, parasitic infection, allergy or cancer includes, but is not limited to, related diseases caused by hepatitis C virus infection, related diseases caused by infection of chlamydia and chlamydia, prostate cancer, breast cancer, Ovarian cancer, lung cancer, gastric cancer, endometrial cancer, salivary gland cancer, adenocarcinoma, colon and rectal cancer, non-small cell lung cancer, lung adenocarcinoma, and the like.
  • the design sequence is as follows - ( 1 ) (G) n (L) n X 1 X 2 CGY 1 Y 2 (M) n (G) n
  • 3 ⁇ 4 A, T or G;
  • L, M A, T, C or G;
  • n is 0-6
  • n is 0-6; , -TCgTAACgTTgTTTTTAACgTT-3 ' (SEQ ID NO: 9) Ggggggggg)Q ( :72TccTTc3S ID NOccET- - ggggggggg) (Q1cEO :7cTcTTS I NTcTDT - ggggggg)ggg (Q0 NO :7cCATSE IDcTcccTClT— - )gggggggggggg (QD NO :69S IcTEccAccAcTcTcTcT3T- - ggggg) (QO :67TTCTCT3SE ID NcAACAAATI - g )gggggg (Q058ccccSED N:cTcTccc3 ITcT— - ggggg) (QO57ccccc3SE IN :TcTDcTcT- -- gggggs (Q"c
  • Gggg ()gggQS7E ID N0:1cccT3ACATAcTTcTcTTC- - gggg)g (QN IDO: 15TCT3SEcTcTcTTcTT_ - gg)gg (QN IDO :14cSECTTTTTTTCTTcTI - gggQ)gg (gS ID N :2T3EO1cTTTcTTcTTTcT— — ggg)g (Qg IED NO10TT3S :CAcATCTCTATAcT- I , -TCgTCgACgTCgTTgggCggg-3 ' (SEQ ID NO:73)
  • DNA synthesis uses a solid phase phosphoramidite triester method to synthesize DNA fragments. This method has the advantages of efficacious and rapid coupling, and has been widely used in DNA chemical synthesis.
  • DNA chemical synthesis differs from the enzymatic DNA synthesis process in that it extends from the 5'-3' direction, but starts at the 3' end.
  • the specific reaction steps are as follows:
  • the protective group DMT (dimethoxytrityl) of the nucleotide attached to CpG (Controlled Pore Glass) was removed with trichloroacetic acid to obtain a free 5'-hydroxyl end for the next condensation reaction. .
  • the phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protection), this intermediate will undergo a condensation reaction with a deprotected nucleotide on GpG.
  • the terminal hydroxyl group is often blocked by acetylation, and the general acetylation reagent is acetic anhydride and N-methyl. Mixture Formed together.
  • the nucleoside monomer is linked to the oligonucleotide attached to CpG through a phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by an acid or a base.
  • a tetrahydrofuran solution of iodine is usually used.
  • the phosphoryl group is converted to a phosphate triester to obtain a stable oligonucleotide.
  • a deoxynucleotide is attached to the nucleotide of C P G, and the deprotected nucleotide of the newly attached deoxynucleotide 5'" is also removed by trichloroacetic acid.
  • the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment.
  • the solid phase synthesis oligonucleotide is carried out on a DNA synthesizer. After the deprotection group is removed from the oligonucleotide synthesized by the above method, the purity of the target oligonucleotide is extremely low, and contains a large amount of impurities, and the main impurities are
  • the removed protecting group is ammonia and benzoic acid ammonia and isobutyric acid ammonia, the nitrile ethyl group removed on the nitrile phosphorus group, and the short chain generated during synthesis, so that the amount of the oligonucleotide in the crude product is only About 15%.
  • the efficiency of each step in the synthesis is between 97% and 98%, the cumulative efficiency is not high. Taking the chain lengths of 20mer and 50mer as examples, (97.5%) 20 ⁇ 60%, (97.5%) 50 ⁇ 28%, it can be seen that the target oligonucleotide content in the crude product is very low, even 10%. To. These impurities, especially the large amounts of salts and short chains present in the crude product, cause quantitative inaccuracy and affect the next reaction, so the oligonucleotide must be purified. Purification by polyacrylamide gel electrophoresis (PAGE) is recommended. The purified product is highly purified and can be used in most molecular biology experiments to avoid many unexpected problems. If cost savings are considered, for less demanding experiments, such as simple PCR reactions, desalting purification can be used.
  • PAGE polyacrylamide gel electrophoresis
  • the oligonucleotide DNA is 0D 26 .
  • the value is measured.
  • Absorbance oligonucleotide solution as defined in 1 0D 26 1, 1cm light path under the standard 1ml quartz cuvette 260nm wavelength. .
  • the base composition is not identical for each particular oligonucleotide, the 1260 0D oligonucleotide DNA weighs approximately 33 g.
  • Example 3 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein hepatitis C virus antigen peptide fusion protein
  • HCV65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HCV multi-epitope HCV core antigen fusion protein
  • Oligo synthetic deoxyoligonucleotide
  • B16 cells HCV+B16 cells
  • Oligo 1 SEQ ID N07
  • Oligo 2 SEQ ID NO106
  • Oligo 3 SEQ ID NO 103
  • Oligo 8 SEQ ID NO 123
  • Oligo 10 SEQ ID NO 159
  • mice 2 ⁇ ⁇ HSP- HCV
  • Oligo group injection 50 ⁇ ⁇ Oligo
  • HSP-HCV + 01igo group injection 20 ⁇ ⁇ HSP- HCV, 50 ⁇ ⁇ 01igo
  • HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo.
  • mice in each group were injected with physiological saline, HSP-HCV application solution, Oligo application solution and HSP-HCV+Oligo mixture.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMM medium containing 10% FBS, and the mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-HCV (20 (g/ml). 37 ° C, 5% C0 2 culture 7 Days.
  • Mouse spleen cells were harvested for effector cells.
  • HCV+B16 cells were cultured in 10% FBS in IMDM medium. The cells were incubated with Cr-labeled HCV+B16 cells (1 hour at 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled HCV+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, each well ⁇ containing 1 ⁇ 10 4 cells. Three effector cells ( ⁇ ) were added at a ratio of 100:1 to the target, and three replicate wells were set. Incubate for 4-6 hours at 37 ° C, 5% C0 2 . The 96-well culture plate was centrifuged for 5 minutes (3000 rpm). The ⁇ supernatant was aspirated from each well and the radioactivity was measured. The cytotoxic activity of effector cells was calculated according to the following formula, expressed as specific killing rate.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the activity of the heat shock protein hepatitis C virus antigen peptide fusion protein by immunologically inducing HCV core antigen-specific cytotoxic T lymphocytes (p ⁇ 0.05). This CTL kills HCV-infected cells in vivo.
  • synthetic single-stranded deoxynucleotides significantly enhance the antiviral (including but not limited to hepatitis C virus) activity of the heat shock protein hepatitis C virus antigen peptide fusion protein.
  • B16 cells transfected with the HCV core antigen multi-epitope antigen peptide gene can be used as a cell model for hepatitis C virus infection (CN02122116. 2).
  • CN02122116. 2 hepatitis C virus infection
  • the injection of synthetic single-stranded deoxynucleotides (Oligo) and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein can stimulate mouse CTL to kill HCV in vivo. Cell activity.
  • HCV65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HCV multi-epitope HCV core antigen fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • HC B16 cells transfection of HCV core antigen B16 cells
  • mice Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-HCV group (20 ⁇ ⁇ HSP-HCV), and Oligo group (injected 50 ⁇ ⁇ Oligo) and HSP-HCV+Oligo group (20 ⁇ ⁇ HSP-HCV, 50 ⁇ ⁇ Oligo).
  • HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo. Each group of mice was injected with normal saline, HSP-HCVs Oligo on days 1, 14, and 21 And HSP- HCV+01igo. At day 24 HCV + B16 cells were seeded (1105 cells / animal), the injection site was the right dorsal skin.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed to take the tumor and weigh the tumor.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of M. tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein to stimulate mouse CTL to kill HCV cells in vivo (p ⁇ 0.05), The performance is as follows: The combined use of synthetic single-stranded oligonucleotides and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein inhibits the growth ability of tumor cells expressing HCV core antigen significantly.
  • Example 4 Synthetic single-stranded deoxynucleotides enhance the specificity of CTL activity induced by the heat shock protein Chlamydia trachomatis major outer membrane protein epitope antigen peptide fusion protein
  • Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein HSP65-Chla
  • HSP65-Chla Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells Chola 16 cells of the major outer membrane protein antigen peptide gene of Chlamydia trachomatis (ATCC, see CN02141977. 9).
  • mice 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group, divided into normal saline group (injected saline), HSP65-Chla group (injected 20 ⁇ ⁇ HSP65-Chla), Oligo group (injected 50 ⁇ ) Oligo), HSP65-Chla +01igo group (injection 2 ( ⁇ g HSP65-Chla, 50 ⁇ ⁇ 01igo).
  • HSP65-Chla and Oligo were prepared in PBS at the required concentrations, and HSP65-Chla+Oligo was injected with HSP65-Chla and Oligo. Groups of mice were injected with saline, HSP65-Chla, Oligo and HSP65_Chla+01igo on days 1, 14, and 21, respectively. Four subcutaneous injections (SC) were performed on the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in the usual manner, and the following procedure was as described in Example 1.
  • Chla+B16 cells were cultured in 10% FBS in IMDM medium. Target cells were seeded with 51 Cr-labeled Chla + B16 cells, and the following procedure was performed as described in Example 1.
  • Synthetic single-stranded deoxynucleotides significantly enhance Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis
  • the outer membrane protein antigen peptide fusion protein was induced to induce the activity of Chlamydia-specific CTL (p ⁇ 0.05). This CTL kills Chlamydia-infected cells in vivo. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein in the treatment and prevention of Chlamydia infection and its related diseases.
  • Example 5 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on Mycobacterium tuberculosis heat shock protein 65 and human prostate specific antigen (PSA) epitope antigen peptide fusion protein 1. Synthetic single Enhancement of inducible specific CTL activity by HSP65-HCV by strand oligonucleotide
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • PSA human prostate specific antigen antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotides
  • B16 cells PSA + B16 cells transfected with the PSA antigen peptide gene (ATCC reference CN01134935. 2).
  • mice Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-PSA group (20 ⁇ ⁇ HSP-PSA), 01 i go group (injection) 50 ⁇ ⁇ 01 i go ), HSP-PSA+Oli go group (20 ⁇ ⁇ HSP-PSA, 50 ⁇ ⁇ 01igo).
  • mice in each group were injected with normal saline, HSP-PSA, Oligo and HSP-PSA+Oli go o in four subcutaneous injections (SC;) in the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP_PSA (20 ( ⁇ g/ml).
  • PSA + B16 cells transfected with the PSA antigen peptide gene were cultured in IMDM medium containing 10% FBS.
  • PSA+B16 cells were labeled with 61 Cr (cultured for 1 hour, 37X, 5% C0 2 ).
  • Use IMDM with 10% FBS Wash the cells three times in total.
  • 51 Cr-labeled PSA 16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of PSK-specific CTLs induced by Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (p ⁇ 0.05). This CTL can kill PSA-expressing tumors. cell. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the heat shock protein PSA antigen peptide fusion protein. Prevention and treatment of the biological activity of prostate cancer. 2. Enhancement of anti-expression of PSA tumor by HSP65-PSA by synthetic single-stranded oligonucleotide
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • PSA human prostate specific antigen antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells PSA + B16 cells transfected with the PSA antigen peptide gene.
  • mice Forty male C57/BL6 mice, 6-8 weeks old, were divided into normal saline group (injected with normal saline), HSP-PSA group (20 ⁇ ⁇ HSP-PSA), and Oligo group (injected 50 ⁇ ⁇ Oligo), HSP-PSA+01igo group (20 g HSP-PSA, 50 ⁇ 01igo).
  • HSP-PSA and Oligo were prepared in PBS, and HSP-PSA+Oligo group was injected with HSP-PSA and Oligo. Mice were injected with saline, HSP-PSA, 01igo and HSP_PSA+01igo on days 1, 14, and 21, respectively. PSA+B16 cells (1 ⁇ 10 5 /piece) were inoculated on the 28th day, and the injection site was subcutaneous in the right back of the mouse.
  • mice On the 15th day after the tumor was inoculated, the mice were sacrificed to take the tumor and weighed the tumor.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (P ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of heat shock proteins and PSA antigen peptide fusion proteins in the prevention and treatment of prostate cancer.
  • Example 6 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein-MUC1 antigen peptide fusion protein 1. Synthesis of specific CTL activity by HSP65-MUC1 induced by synthetic single-stranded oligonucleotide Enhancement
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-MUC1 antigen peptide fusion protein HSP-MUC1 antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotides
  • B16 cells MUC1+B16 cells transfected with the WJC1 antigen peptide gene (ATCC reference CN01102614.6).
  • mice 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group were divided into normal saline group (injected saline), HSP-MUC1 group (20 ⁇ ⁇ HSP-MUCl injection), Oligo group (injected 50 ⁇ 6) Oligo), HSP- MUCl+Oligo group (20 ⁇ ⁇ HSP-MUCl, 50 ⁇ ⁇ 01igo).
  • HSP-MUCl and Oligo were prepared in PBS to the desired concentration, and the HSP-MUCl+Oligo group was injected with HSP-MUC1 and Oligo.
  • Each group of mice was injected with physiological saline, HSP-MUC1, Oligo and HSP-MUCl+01igo on days 1, 14, and 21.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-MUC1 (200 g/ral). Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
  • MUC1+B16 cells were cultured in 10% FBS in IMDM medium.
  • PSA+B16 cells were labeled with 51 Cr (1 hour, 37 ° C, 5% C0 2 ) o
  • the cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled UC1+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein to induce MUC1-specific cytotoxic T lymphocytes (p ⁇ 0.05), this CTL can kill expression Tumor cells of UC1. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the prevention and treatment of MUC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. by the Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein. Learning activity.
  • the synthetic single-stranded oligonucleotide enhances the anti-expression of MUC1 tumor activity by HSP65-MUC1.
  • mice Female C57/BL6 mice were used for 6-8 weeks, divided into normal saline group (injected saline), HSP-MUC1 group (20 ⁇ ⁇ HSP-MUC1 injection), Oligo group (injected 50 ⁇ ⁇ Oligo), HSP-MUCl+Oligo group (20 ⁇ ⁇ HSP-MUC1, 50 ⁇ ⁇ Oligo), 10 in each group.
  • MUC1+B16 cells (I x lO 5 /only) were inoculated on the 28th day, and the injection site was subcutaneously in the right hind back of the mouse.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of MUC1 tumor cells inhibited by Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein (p ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein for the prevention and treatment of UC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. .
  • Example 7 Enhancement of immunostimulatory effects of synthetic single-stranded deoxynucleotides on heat shock protein HER2 antigen peptide fusion protein
  • HSP-HER-2 antigen peptide fusion protein HSP-HER-2
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells HER- 2+B16 cells transfected with the HER-2 antigen peptide gene (ATCC, see CN01136347. 9).
  • mice 6-8 weeks of female C57/BL6 mice were divided into normal saline group (injected saline), HSP-HER-2 group (20 ⁇ ⁇ HSP-HER - 2 injection), Oligo group (50 ⁇ Oligo injection), HSP - HER- 2+01 i go group (injected 20 g HSP-HER-2, 50 g Oligo), 10 in each group.
  • HSP-HER-2 and Oligo were formulated in PBS, and HSP-HER-2+01igo was prepared by mixing HSP-HER-2 with Oligo.
  • mice were injected with saline, HSP-HER-2, Oligo, and HSP-HER-2+OligOo at four subcutaneous injections (SC) in the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • IMDM medium containing 10% FBS
  • cells were resuspended, IMDM medium containing 10% ConA and HSP- HER-2 (200 ⁇ ⁇ ) stimulation of mouse spleen cells was added.
  • Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
  • B16 cells (HER-216 cells) transfected with the HER-2 antigen peptide gene were cultured in 10% FBS in IMDM medium.
  • HER-2+B16 cells were labeled with 51 Cr (cultured for 1 hour, 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled 1 ⁇ -2 16 cells were added to 10% FBS in IMDM medium to wells of a 96-well culture plate, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a target ratio of 100:1, and three replicate wells were set.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein and HER-2 antigen peptide fusion protein to induce HER-2 specific cytotoxic T lymphocytes (p ⁇ 0.05). CTL can kill tumor cells expressing HER-2. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer. Second, the synthetic single-stranded oligonucleotide enhances the anti-expression of HSP65-HER-2 by HER-2 tumor activity
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HER-2 HER-2 antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • HER-2+B16 cells transfected HER-2 B16 cells of the antigenic peptide gene.
  • HSP-HER-2 injected with 20 ⁇ 8 HSP-HER-2
  • Oligo group injected with 50 ⁇ ⁇ Oligo.
  • HSP-HER- 2+01 i go group (20 ⁇ ⁇ HSP-HER- 2, 50 ⁇ Oligo), 10 in each group.
  • mice were injected with saline, HSP-HER-2, Oligo and HSP-HER-2+01igo on days 1, 14, and 21, respectively.
  • HER-2+B16 cells (1 ⁇ 10 5 /piece) were inoculated, and the injection site was subcutaneous in the right back of the mouse.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins (P ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock egg S 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer.

Abstract

The present invention provides a kind of single strand deoxynucleotide containing CpG capable of reinforcing the immunostimulation effect of antigen or antigen composition, vaccine composition consisting of said single strand deoxynucleotide and antigen or antigen composition, and method of preparing the vaccine. The single strand deoxynucleotide containing CpG significantly enhance the activity of heat shock protein-antigen peptide fusion protein to induce antigen specific CTL, and antigen specific anti-tumor, anti-virus and anti-chlamydia infection. Moreover, the present invention provides the use of single strand deoxynucleotide containing CpG in preparing vaccine composition and method of enhancing immunostimulation effect, said vaccine composition can be used as drug to treat virus infection, bacteria infection, parasitic infection allergy or cancer.

Description

人工合成的单链脱氧核苷酸与其疫苗组合物及其应用 技术领域  Synthetic single-stranded deoxynucleotide and vaccine composition thereof and application thereof
本发明涉及一种能够增强抗原或抗原组合物免疫刺激作用的人工合成的单链脱 氧核苷酸， 以及由所述单链脱氧核苷酸与抗原或抗原组合物所组成疫苗组合物。此外， 本发明还涉及人工合成的单链脱氧核苷酸在制备疫苗组合物中的应用， 以及所述疫苗 组合物作为药物的应用。 背景技术  The present invention relates to a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory action of an antigen or antigen composition, and a vaccine composition comprising the single-stranded deoxynucleotide and an antigen or antigen composition. Furthermore, the invention relates to the use of synthetic single-stranded deoxynucleotides for the preparation of vaccine compositions, and to the use of said vaccine compositions as medicaments. Background technique
1984年， Tokunaga T等发现卡介苗 （BCG) DNA能抑制肿瘤的生长， 激活人外周血 单个核细胞和天然杀伤 （NK) 细胞， 诱生干扰素 （IFN)， 这些功能依赖于 DNA分子中  In 1984, Tokunaga T et al. found that BCG DNA inhibited tumor growth, activated human peripheral blood mononuclear cells and natural killer (NK) cells, and induced interferon (IFN). These functions depend on DNA molecules.
Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity, JNCI , 72 : 955. 1984)。 CpG是由胞嘧啶和鸟嘌呤通过磷酸连接 成的二核苷酸， 其中 C代表胞嘧啶， G代表鸟嘌呤， p代表磷酸， 胞嘧啶位于 5' 端。 近 年来的研究表明， 人工合成的含一个或多个 CpG的寡核苷酸单链 DNA (CpG 0DN) 也可 表现免疫调节作用， 可增强 B细胞、 T细胞、 NK细胞、 抗原提呈细胞 （如单核细胞、 巨 噬细胞和树突状细胞） 和中性粒细胞的活性， 并表现出明显的临床应用前景 (Weiner GJ， The iramunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol 2000 Oct ; 68 (4) : 455-63)。 但由于序列的 不同， 人工合成的单链寡核苷酸可有多种多样的形式。 Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity, JNCI, 72: 955. 1984). CpG is a dinucleotide in which cytosine and guanine are linked by a phosphate, wherein C represents cytosine, G represents guanine, p represents phosphoric acid, and cytosine is located at the 5' end. Recent studies have shown that artificially synthesized single-stranded DNA (CpG 0DN) containing one or more CpGs can also exhibit immunomodulatory effects, enhancing B cells, T cells, NK cells, and antigen presenting cells ( Such as monocytes, macrophages and dendritic cells) and neutrophil activity, and showed significant clinical application prospects (Weiner GJ, The iramunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol 2000 Oct; 68 (4) : 455-63). However, synthetic single-stranded oligonucleotides can take a wide variety of forms due to the different sequences.
热休克蛋白 （heat shock protein, HSP) 是存在于多种生物体内的一个分子伴 侣蛋白质家族。 在免疫应答的过程中， 热休克蛋白可表现出如下四种基本的功能： 1、 携带抗原进入包括树突状细胞在内的抗原提呈细胞； 2、 引导抗原在抗原提呈细胞内 进入 MHC I 类途径加工提呈； 3、 刺激包括树突状细胞在内的抗原提呈细胞表达协同 刺激分子， 分泌细胞因子； 4、 使携带抗原与热休克蛋白形成复合物或融合蛋白的抗 原， 以获得激活抗原特异性细胞毒性 T淋巴细胞的性质 (Tamura Y, Peng P, Liu K， Daou M， Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997 Oct 3 ; 278 (5335) : 117-20 )。  Heat shock protein (HSP) is a family of molecular companion proteins found in many organisms. In the process of immune response, heat shock proteins can exhibit the following four basic functions: 1. Carrying antigens into antigen-presenting cells including dendritic cells; 2. Directing antigens into antigen-presenting cells into MHC Class I pathway processing and presentation; 3. Stimulation of antigen-presenting cells including dendritic cells to express costimulatory molecules, secreting cytokines; 4. Antigens that bind antigens and heat shock proteins to form complexes or fusion proteins, Obtaining the property of activating antigen-specific cytotoxic T lymphocytes (Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997 Oct 3 ; 278 (5335) : 117-20 ).
] 细胞毒性 T淋巴细胞 （cytotoxic T lymphocyte, CTL) 是一个最强力的个体杀 伤肿瘤细胞和杀伤病毒感染细胞的免疫细胞。 外源性的蛋白类抗原在应用个体后， 在 包括树突状细胞在内的抗原提呈细胞中主要经 MHC II类途径加工提呈， 激发体液免 疫反应 (Heikema A， Agsteribbe E, Wilschut J, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1 ; 57 (1-3) : 69-74) , 不能有效地诱生抗原特异性 CTL o ] Cytotoxic T lymphocyte (CTL) is one of the most powerful individuals to kill tumor cells and kill cells infected with virus-infected cells. Exogenous protein antigens are mainly processed by MHC class II pathway in antigen-presenting cells including dendritic cells after application of the individual, and stimulate humoral immune response (Heikema A, Agsteribbe E, Wilschut J, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1 ; 57 (1-3) : 69-74), can not effectively induce antigen-specific CTL o
分枝杆菌热休克蛋白 65能够携带与其融合的或结合的或偶联的抗原肽进入 MHC I 类抗原提呈途径， 激活抗原肽特异性的细胞毒性 T淋巴细胞， 杀伤表达抗原肽的肿瘤 细胞。  Mycobacterial heat shock protein 65 is capable of carrying an antigenic peptide fused or conjugated or coupled thereto into the MHC class I antigen-presenting pathway, activating antigen-peptide-specific cytotoxic T lymphocytes, and killing tumor cells expressing the antigen peptide.
丙型肝炎病毒 （HCV) 是引起丙型肝炎的病原体， 在全世界感染了大约一亿七千 多万人。 肝癌和肝硬化是 HCV感染引起的两个严重的并发症。 在世界范围内， 采用 α -干扰素和病毒唑联合应用是治疗丙型肝炎病毒感染的方法， 其有效率约为 40% (Jon Cohen. The Scientific Challenge of Hepatitis C. Science, 1999, 285 : 26 - 30)。 采用结核杆菌热休克蛋白 65 (HSP65) 与多表位 HCV核心抗原相融合所形成的融合蛋 白是一种对丙型肝炎有预防和治疗作用的基因工程重组蛋白疫苗 （CN02122116. 2, 中 国）。 '  Hepatitis C virus (HCV) is the causative agent of hepatitis C and has infected more than 170 million people worldwide. Liver cancer and cirrhosis are two serious complications caused by HCV infection. Worldwide, the combination of alpha-interferon and ribavirin is a method of treating hepatitis C virus infection with an effective rate of approximately 40% (Jon Cohen. The Scientific Challenge of Hepatitis C. Science, 1999, 285: 26) - 30). The fusion protein formed by the fusion of Mycobacterium tuberculosis heat shock protein 65 (HSP65) and the multi-epitope HCV core antigen is a genetically engineered recombinant protein vaccine for preventing and treating hepatitis C (CN02122116. 2, China). '
沙眼衣原体 （Chlamydia Trachomatis, CT) 是不运动的专营细胞内寄生的微生 物， 可在人类引起沙眼、 包涵体性结膜炎、 泌尿生殖系统疾病、 性病淋巴肉芽肿、 子 宫内膜炎、 输卵管炎、 盆腔炎、 输卵管性不孕及输卵管异位妊娠。  Chlamydia Trachomatis (CT) is a non-sports bacterium that specializes in intracellular parasitic microorganisms, which can cause trachoma, inclusion body conjunctivitis, genitourinary diseases, sexually transmitted diseases, lymphogranuloma, endometritis, salpingitis, pelvic cavity in humans. Inflammation, tubal infertility and ectopic pregnancy of the fallopian tube.
沙眼衣原体的主要外膜蛋白 （M0MP) 可剌激保护性免疫。 釆用结核杆菌热休克蛋 白 65和沙眼衣原体主要外膜蛋白表位抗原肽相融合所形成的融合蛋白是一种对人沙 眼衣原体感染及其相关疾病有预防和治疗作用的重组蛋白 （CN02141977. 9, 中国） 。  The major outer membrane protein (M0MP) of Chlamydia trachomatis stimulates protective immunity. The fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein epitope antigen peptide is a recombinant protein which has preventive and therapeutic effects on human Chlamydia trachomatis infection and related diseases (CN02141977. 9) , China).
人前列腺特异性抗原 （Prostate specific antigen, PSA) 是表达在前列腺癌 细胞的一种组织特异性抗原。 采用结核杆菌热休克蛋白 65和 PSA表位抗原肽相融合所 形成的融合蛋白是一种对人前列腺癌有预防和治疗作用的重组蛋白 （CN01134935. 2， 中国） 。  Human Prostate specific antigen (PSA) is a tissue-specific antigen expressed in prostate cancer cells. The fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and PSA epitope antigen peptide is a recombinant protein for preventing and treating human prostate cancer (CN01134935. 2, China).
MUC1蛋白是粘蛋白 （mucins) 的一种， 高水平表达在乳腺癌、 卵巢癌、 肺癌、 前列腺癌、 结直肠癌细胞， 为免疫治疗的靶点。 采用结核杆菌热休克蛋白 65和 MUC1 抗原肽相融合所形成的融合蛋白是一种对表达 MUC1 蛋白的肿瘤 （包括但不限于乳腺 癌） 具有预防和治疗作用的重组蛋白 （CN01.102614 6, 中国） 。 The MUC1 protein is a type of mucins that are expressed at high levels in breast cancer, ovarian cancer, lung cancer, prostate cancer, and colorectal cancer cells, and are targets for immunotherapy. The fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and MUC1 antigen peptide is a tumor that expresses MUC1 protein (including but not limited to breast) Cancer) Recombinant protein with preventive and therapeutic effects (CN01.102614 6, China).
HER - 2蛋白 （HER - 2) 是一种和表皮生长因子受体 （Tadashi Yamamoto, et al. Similarity of protein encoded by the human c - erbB-2 gene to epidermal growth factor receptor. Nature vol 319 16 January 1986, 230-234) 具有很高同源性 的跨膜蛋白， 在 30%的人乳腺肿瘤细胞中过度表达， 是乳腺癌免疫治疗的靶点。 采用 结核杆菌热休克蛋白 65和 HER-2抗原肽相融合所形成的融合蛋白是一种对人乳腺癌 具有预防和治疗作用的重组蛋白 （CN01136347. 9, 中国） 。  HER - 2 protein (HER - 2) is an epidermal growth factor receptor (Tadashi Yamamoto, et al. Similarity of protein encoded by the human c - erbB-2 gene to epidermal growth factor receptor. Nature vol 319 16 January 1986 , 230-234) Transmembrane proteins with high homology, overexpressed in 30% of human breast tumor cells, are targets for breast cancer immunotherapy. The fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide is a recombinant protein that has preventive and therapeutic effects on human breast cancer (CN01136347. 9, China).
上述研究结果表明， 将人工合成的单链寡核苷酸与热休克蛋白相融合的抗原或抗 原组合物联合应用具有广阔的前景。 然而， 截至到本发明为止， 未见相关的文献报道 或公幵。 发明内容  The results of the above studies indicate that the combination of an artificially synthesized single-stranded oligonucleotide and an antigen or antigen composition fused with a heat shock protein has broad prospects. However, as of the present invention, no relevant literature reports or publications have been found. Summary of the invention
本发明的目的之一是提供能够增强抗原或抗原组合物免疫剌激作用的含人工合 成的单链脱氧核苷酸， 其可作为抗原或抗原组合物的佐剂来增强抗原或抗原组合物的 免疫剌激作用。 它们由含一个或多个人工合成的寡核苷酸单链 DNA分子构成， 其磷酸 二酯键可以是部分硫化的， 全部硫化的， 也可以是未硫化的。 本发明的人工合成的单链脱氧核苷酸包含至少一个选自下式 (1) - (5)任一项的序 列：  One of the objects of the present invention is to provide a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory effect of an antigen or antigen composition, which can be used as an adjuvant for an antigen or antigen composition to enhance an antigen or antigen composition. Immune stimulation. They consist of single-stranded DNA molecules containing one or more synthetic oligonucleotides, which may be partially sulfurized, fully vulcanized, or unvulcanized. The artificially synthesized single-stranded deoxynucleotide of the present invention comprises at least one sequence selected from any one of the following formulas (1) to (5):
( 1 ) (G)„(L) n ΧΜΥ,Υ^Μ) » (G)„， 其中 X^ A， T或 G; 为 A或 T; Υ^ Α或 Τ; Υ2 为 ， Τ或 C; L和 Μ分别为 Α， Τ, C或 G; η为 0-6的整数； 优选地， 式 （1 ) 中人工合成的单链脱氧核苷酸具有选自下列任一所示的序列：(1) (G)„(L) n ΧΜΥ,Υ^Μ) » (G)„, where X^ A, T or G; is A or T; Υ^ Α or Τ; Υ 2 is, Τ or C ; L and Μ are Α, Τ, C or G; η is an integer of 0-6; single-chain deoxy preferably, the formula (1) in synthetic nucleotides having a sequence selected from any of the following is shown:
5' -ggggTCgTTCgTCgTTgggggg-3 ' (SEQ ID NO : 2) 5' -ggggTCgTTCgTCgTTgggggg-3 ' (SEQ ID NO : 2)
5， -ggggATAACgTTgCgggggg-3 ' (SEQ ID NO : 3)  5, -ggggATAACgTTgCgggggg-3 ' (SEQ ID NO : 3)
5， -ggggTgCAACgTTCAgggggg-3 ' (SEQ ID NO :4)  5, -ggggTgCAACgTTCAgggggg-3 ' (SEQ ID NO : 4)
5， -ggggTCCTACgTAggAgggggg-3 ' (SEQ ID NO :8)  5, -ggggTCCTACgTAggAgggggg-3 ' (SEQ ID NO : 8)
5， - ggggTCCATgACgTTCCTgAAgggggg- 3， (SEQ ID NO : 23)  5, - ggggTCCATgACgTTCCTgAAgggggg- 3, (SEQ ID NO : 23)
5， -gggggACgTCgCCggggggg-3 ' (SEQ ID NO : 37)  5, -gggggACgTCgCCggggggg-3 ' (SEQ ID NO : 37)
5' -ggATCCgTACgCATgggggg-3 ' (SEQ ID NO : 38)  5' -ggATCCgTACgCATgggggg-3 ' (SEQ ID NO : 38)
5' -gggggAATCgATTCgggggg-3' (SEQ ID NO: 101) )' -gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 100) 5'-gggggAATCgATTCgggggg-3' (SEQ ID NO: 101) ) '-gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 100)
)' -ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 102)  ) - -ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 102)
Ϋ -gggACgTACgTCgggggg-3' (SEQ ID NO : 105)  Ϋ -gggACgTACgTCgggggg-3' (SEQ ID NO: 105)
5' -gggggATCgACgTCgATCgggggg-3， (SEQ ID NO： 108)  5' -gggggATCgACgTCgATCgggggg-3, (SEQ ID NO: 108)
Ϋ -ggCgATCgATCgATCggggggg-3' (SEQ ID NO : 111)  Ϋ -ggCgATCgATCgATCggggggg-3' (SEQ ID NO : 111)
i' -ggggTCgATCgATCgAgggggg-3' (SEQ ID NO: 112)  i' -ggggTCgATCgATCgAgggggg-3' (SEQ ID NO: 112)
；， -ggTCgCgATCgCgAgggggg-3 ' (SEQ ID NO: 114)  ;, -ggTCgCgATCgCgAgggggg-3 ' (SEQ ID NO: 114)
；， -ggGGTCAACGTTGAgggggG-3 ' (SEQ ID NO : 128)  ;, -ggGGTCAACGTTGAgggggG-3 ' (SEQ ID NO : 128)
；' -gTCgTTTTCgTCgACgAATTgggggggg-3 ' (SEQ ID NO : 164)  ;' -gTCgTTTTCgTCgACgAATTgggggggg-3 ' (SEQ ID NO : 164)
；， -gTCgTTATCgTTTTTTCgTAgggggg-3 ' (SEQ ID NO : 165) ;, - g TCgTTATCgTTTTTTCgTAgggggg-3 ' (SEQ ID NO : 165)
；' -ggCgTTAACgACgggggg-3' (SEQ ID NO : 166)  ;' -ggCgTTAACgACgggggg-3' (SEQ ID NO : 166)
；' -gTCggCACgCgACgggggg-3' (SEQ ID NO : 52)  ;' -gTCggCACgCgACgggggg-3' (SEQ ID NO : 52)
；， -ggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 160)  ;, -ggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 160)
；，— gTCTATTTTgTACgTACgTgggg- 3， (SEQ ID NO : 169)  ;, — gTCTATTTTgTACgTACgTgggg- 3, (SEQ ID NO : 169)
；， -gACgTCgACgTCgACgTCAggggg-3 ' (SEQ ID NO : 170)  ;, -gACgTCgACgTCgACgTCAggggg-3 ' (SEQ ID NO : 170)
；' -ggggTCgATCgTTgCTAgCgggggg-3 ' (SEQ ID NO : 173)  ;' -ggggTCgATCgTTgCTAgCgggggg-3 ' (SEQ ID NO : 173)
；， -gggggACgTTATCgTATTggggggg-3 ' (SEQ ID NO : 174)  ;, -gggggACgTTATCgTATTggggggg-3 ' (SEQ ID NO : 174)
；， -ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3' (SEQ ID NO: 175)  ;, -ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3' (SEQ ID NO: 175)
5， -ACgATCgATCgATCgggggg-3 ' (SEQ ID NO : 180)  5, -ACgATCgATCgATCgggggg-3 ' (SEQ ID NO : 180)
5， -AgACgTCTAACgTCggggg-3 ' (SEQ ID NO : 168)  5, -AgACgTCTAACgTCggggg-3 ' (SEQ ID NO : 168)
5， -ggggTgCTggCCgTCgTTgggggg-3 ' (SEQ ID NO : 5)  5, -ggggTgCTggCCgTCgTTgggggg-3 ' (SEQ ID NO : 5)
5， -ggggTCgTTgCCgTCgggggg-3 ' (SEQ ID NO : 6)  5, -ggggTCgTTgCCgTCgggggg-3 ' (SEQ ID NO : 6)
5， -ACCggTATCgATgCCggTgggggg-3 ' (SEQ ID NO : 22)  5, -ACCggTATCgATgCCggTgggggg-3 ' (SEQ ID NO : 22)
5， -TTCgTTgCATCgATgCATCgTTgggggg-3 ' (SEQ ID NO :28)  5, -TTCgTTgCATCgATgCATCgTTgggggg-3 ' (SEQ ID NO : 28)
(2) (G) n (L) nCG (XY)„CG (M) n (G) n, 其中 X为 A或 T; Y为 A或 T; L和 M分别为 A， T， C或 G; n为 0-6的整数； 优选地， 式 （2) 中人工合成的单链脱氧核苷酸具有选自下列任一所示的序列： 5， -ggggACgATACgTCggggggg-3 ' (SEQ ID N0 : 1) (2) (G) n (L) n CG (XY) „ CG (M) n (G) n , where X is A or T; Y is A or T; L and M are A, T, C or G; n is an integer from 0 to 6; preferably, the artificially synthesized single-stranded deoxynucleotide of formula (2) has a sequence selected from any one of the following: 5, -ggggACgATACgTCggggggg-3 ' (SEQ ID N0: 1)
5' -ggggACgATATCgATgggggg-3 ' (SEQ ID NO : 7)  5' -ggggACgATATCgATgggggg-3 ' (SEQ ID NO : 7)
5' -ggACgATCgATCgTgggggg-3' (SEQ ID NO : 99) ggggggg ()QCTC3AATAATA3TTS ID7cE NO1 :- - cn gggggggg) (QccATcAATA36TTS ID N1cEO : In i n cn n n tn cn ai n n oi cn n n n m cji n tn n cn n cn rri oi ai cn ri ggg¾g)g (QT?27TTcTcTcTO :1TcT3E I NcASD - gggg) (QC3ACTCTCTC I118ATcTTTSED N :CTO- - gggg ()Q31ACTCTCcS ID 17TCAATTCE NOTC:I —5'-ggACgATCgATCgTgggggg-3' (SEQ ID NO: 99) Ggggggg ()QCTC3AATAATA3TTS ID7cE NO1 :- - cn gggggggg) (QccATcAATA36TTS ID N1cEO : In in cn nn tn cn ai nn oi nn nnnm cji n tn n cn n cn rri oi ai cn ri ggg3⁄4g)g (QT?27TTcTcTcTO :1TcT3E I NcASD - gggg) (QC3ACTCTCTC I118ATcTTTSED N : CTO- - gggg ()Q31ACTCTCcS ID 17TCAATTCE NOTC:I —
ggggggggg) (Qc!r93cATCAS ID :cccAcE NOc- — Ggggggggg) (Qc!r93cATCAS ID :cccAcE NOc- —
ggggggggg) (QrccTccTlSE N01TTcA ID:9l — Ggggggggg) (QrccTccTlSE N01TTcA ID: 9l -
gggggg) (Qcc3 :90CCTcccccS IOTATAcED NA- — Gggggg) (Qcc3 : 90CCTcccccS IOTATAcED NA- —
ggggg (Q)SCE I :8TCccTTTc3D NO9Tc- — Ggggg (Q)SCE I :8TCccTTTc3D NO9Tc- —
ggggggggg) (Q8 NO :8cCTCCATc3EcTTAATCS ID- l Ggggggggg) (Q8 NO : 8cCTCCATc3EcTTAATCS ID- l
ggggggg) (Q0:76TTMCTTTccccSE ID NAcc - ggggggg) (Qcc NO :75TTMCcc3S IDTTACCTTEl — Ggggggg) (Q0:76TTMCTTTccccSE ID NAcc - ggggggg) (Qcc NO :75TTMCcc3S IDTTACCTTEl —
gggg (Q)SE I NO8cccTAcTT3D :6Tc— — Gggg (Q)SE I NO8cccTAcTT3D :6Tc—
gggggg ()Q3S53cAccAccccccED NO :Tcc I— -、 Gggggg ()Q3S53cAccAccccccED NO :Tcc I--,
gggggg (Q)AcSE ID NO: 51TcccAc3 l- ggggg (Q)E00TcccTS ID N:5TcTT3 -— Gggggg (Q)AcSE ID NO: 51TcccAc3 l- ggggg (Q)E00TcccTS ID N:5TcTT3 -—
gggggg) (Q93SED4TTccTcc I NO :AccTccc— - gggggg) (Q3S ID:48ACAACcAMTACTE N0TC- — Gggggg) (Q93SED4TTccTcc I NO :AccTccc— - gggggg) (Q3S ID:48ACAACcAMTACTE N0TC- —
ggggg ()Q3SE ID NO :46ACAACcAAATACCT— —、 Ggggg ()Q3SE ID NO :46ACAACcAAATACCT— —,
ggggggggggg ()Q :A3SE ID NO44cccAAAAccAAcAc- - gggg) (Q9cE NO :2TCTTcTT3S IDTCcTcTTCTTCT- I Ggggggggggg ()Q : A3SE ID NO44cccAAAAccAAcAc- - gggg) (Q9cE NO :2TCTTcTT3S IDTCcTcTTCTTCT- I
gggg) (QS I NO :16cTTCCTT3EDTTTTcTI I Gggg) (QS I NO :16cTTCCTT3EDTTTTcTI I
ggggggggggggg) (Q3S IDO: 172TAACE NTccTT— — Ggggggggggggg) (Q3S IDO: 172TAACE NTccTT —
gggggggggggggg)g (Q: 173S ID NO1TcATCEAT- l Gggggggggggggg)g (Q: 173S ID NO1TcATCEAT- l
gggggggggggg ()Q3S ID NO :113cATATCETCcA- —- ggggggggggg)g (Q IDO :1043SE NTCTCTACATCAA- —- gggggggggg)gg (Q IDO :103SE N9CACATATTATCC- —- ggggggggg)gg (Q103SD NO :1E IATCTCATCATA- — Gggggggggggg ()Q3S ID NO :113cATATCETCcA- --- ggggggggggg)g (Q IDO :1043SE NTCTCTACATCAA- --- gggggggggg)gg (Q IDO :103SE N9CACATATTATCC- --- ggggggggg)gg (Q103SD NO :1E IATCTCATCATA- —
ggggggggggg)g (Qg : IDO1073SE NATCACACATTT- —- ggggggggggg)g (Q63 NO :10TSE IDTTCcccAA- -- 5， -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO :143) Ggggggggggg)g (Qg : IDO1073SE NATCACACATTT- --- ggggggggggg)g (Q63 NO :10TSE IDTTCcccAA- -- 5, -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 143)
5， -actcgagacgcccgttgatagctt-3' (SEQ ID NO: 145)  5, -actcgagacgcccgttgatagctt-3' (SEQ ID NO: 145)
5， - AACgTTggCgTCgACgTCAgCgCC- 3， (SEQ ID NO :146)  5, - AACgTTggCgTCgACgTCAgCgCC-3, (SEQ ID NO: 146)
5， -gACgTCgACgTTgACgCT-3 ' (SEQ ID NO :147) 5, - g ACgTCgACgTTgACgCT-3 ' (SEQ ID NO : 147)
5， - ggCgTTAACgTTAgCgCT- 3， (SEQ ID NO :148)  5, - ggCgTTAACgTTAgCgCT- 3, (SEQ ID NO: 148)
5， -AgCgCTAgCgCTgACgTT-3' (SEQ ID NO :149)  5, -AgCgCTAgCgCTgACgTT-3' (SEQ ID NO: 149)
5 ' -CTAgACgTTCAAgCgTT-3， (SEQ ID NO: 150)  5 ' -CTAgACgTTCAAgCgTT-3, (SEQ ID NO: 150)
5， - gACgATCgTCgACgATCgTC - 3， (SEQ ID NO: 156)  5, - gACgATCgTCgACgATCgTC - 3, (SEQ ID NO: 156)
5， -gTCgTTCgTAgTCgACTACgAgTT-3 ' (SEQ ID NO: 161)  5, -gTCgTTCgTAgTCgACTACgAgTT-3 ' (SEQ ID NO: 161)
5， -AAAAgACgTCgACgTCgACgTCTTTT- 3， (SEQ ID NO :162)  5, -AAAAgACgTCgACgTCgACgTCTTTT- 3, (SEQ ID NO: 162)
5， -TgCgACgATCgTCgCACgATCggAT-3 ' (SEQ ID NO :176)  5, -TgCgACgATCgTCgCACgATCggAT-3 ' (SEQ ID NO: 176)
5， -TgCgACgTCgCACAgCgT-3 ' (SEQ ID NO :177)  5, -TgCgACgTCgCACAgCgT-3 ' (SEQ ID NO: 177)
(3) (TCG)„(L)nCG (M)n(G)„， 其中 L和 M分别为 A， T, C或 G; n为 0-6的整数； 优选地， 式（3) 中人工合成的单链脱氧核苷酸具有选自下列任一所示的序列:(3) (TCG) „(L) n CG (M) n (G)„, where L and M are respectively A, T, C or G; n is an integer from 0 to 6; preferably, formula (3) The synthetic single-stranded deoxynucleotide has a sequence selected from any one of the following:
5， -TCgTTgCCgTCgg-3' (SEQ ID NO :59) 5, -TCgTTgCCgTCgg-3' (SEQ ID NO: 59)
5， - TCgTTgCCgTCggg- 3， (SEQ ID NO :60)  5, - TCgTTgCCgTCggg-3, (SEQ ID NO: 60)
5， -TCgTTgCCgTCgggg- 3， (SEQ ID NO :61)  5, -TCgTTgCCgTCgggg- 3, (SEQ ID NO: 61)
5， -TCgTTgCCgTCggggg-3' (SEQ ID NO :62)  5, -TCgTTgCCgTCggggg-3' (SEQ ID NO: 62)
5， -TCgTTgCCgTCgggggg-3' (SEQ ID NO :63)  5, -TCgTTgCCgTCgggggg-3' (SEQ ID NO: 63)
5， -TCgTTgCCgTCggggggg-3' (SEQ ID NO :64)  5, -TCgTTgCCgTCggggggg-3' (SEQ ID NO: 64)
5， -TCgTTgCCgTCgggggggg-3' (SEQ ID NO :65)  5, -TCgTTgCCgTCgggggggg-3' (SEQ ID NO: 65)
5， - TCgTTgCCgTCggggggggg- 3， (SEQ ID NO :66)  5, - TCgTTgCCgTCggggggggg- 3, (SEQ ID NO: 66)
5' -TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO: 103)  5' -TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO: 103)
5， - TCgTCgggTgCAACgTTgCAgggggg- 3， (SEQ ID NO :129)  5, - TCgTCgggTgCAACgTTgCAgggggg-3, (SEQ ID NO: 129)
5， -TCgTCgggTgCgTCgACgCAgggggg-3 ' (SEQ ID NO :130)  5, -TCgTCgggTgCgTCgACgCAgggggg-3 ' (SEQ ID NO: 130)
5， - TCgTCgggTgCgATCgCAgggggg- 3' (SEQ ID NO: 131)  5, - TCgTCgggTgCgATCgCAgggggg- 3' (SEQ ID NO: 131)
5， -TCgTCgggTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO :132)  5, -TCgTCgggTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO: 132)
5， -TCgTCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 133)  5, -TCgTCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 133)
5， -TCgTCgCAgAACgTTCTgggggg-3 ' (SEQ ID NO :134)  5, -TCgTCgCAgAACgTTCTgggggg-3 ' (SEQ ID NO: 134)
5， -TCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO :135) 5， -TCgTgCgACgATCgTCgCAgggggg-3 ' (SEQ ID NO: 139) 5, -TCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 135) 5, -TCgTgCgACgATCgTCgCAgggggg-3 ' (SEQ ID NO: 139)
5， -TCgTATgCATCgATgCATAgggAgg-3 ' (SEQ ID NO :140)  5, -TCgTATgCATCgATgCATAgggAgg-3 ' (SEQ ID NO :140)
5， -TCgTgCATCgATgCAgggggg-3 ' (SEQ ID NO :157)  5, -TCgTgCATCgATgCAgggggg-3 ' (SEQ ID NO: 157)
5， -TCgAAACgTTTCgggggg-3 ' (SEQ ID NO :158)  5, -TCgAAACgTTTCgggggg-3 ' (SEQ ID NO :158)
5， -TCggACgATCgTCgggggg-3' (SEQ ID NO :159)  5, -TCggACgATCgTCgggggg-3' (SEQ ID NO: 159)
5， -TCgAgCgATCgCTCgAgggggg-3 ' (SEQ ID NO: 179)  5, -TCgAgCgATCgCTCgAgggggg-3 ' (SEQ ID NO: 179)
5， -TCgTCgCTTTgTCgTTgggg-3 ' (SEQ ID NO: 13)  5, -TCgTCgCTTTgTCgTTgggg-3 ' (SEQ ID NO: 13)
5， - TCgTCgTTTTgTCgTTgggg- 3， (SEQ ID NO :11)  5, - TCgTCgTTTTgTCgTTgggg- 3, (SEQ ID NO: 11)
5， -TCgTCgggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO :18)  5, -TCgTCgggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 18)
5， -TCgTCgggTgCgACgATCgTCgggggg-3 ' (SEQ ID NO: 19)  5, -TCgTCgggTgCgACgATCgTCgggggg-3 ' (SEQ ID NO: 19)
5， - TCgTCgTTTgCATCgATgCAggggggg- 3， (SEQ ID NO :21)  5, - TCgTCgTTTgCATCgATgCAggggggg- 3, (SEQ ID NO: 21)
5' -TCgTCgTTTTgACgATCgTCgggggg-3 ' (SEQ ID NO :24)  5' -TCgTCgTTTTgACgATCgTCgggggg-3 ' (SEQ ID NO :24)
5， - TCgTTCggggTgCCg- 3， (SEQ ID NO :80)  5, - TCgTTCggggTgCCg- 3, (SEQ ID NO: 80)
5， - TCgTTCggggTACCgATgggg- 3， (SEQ ID NO :84)  5, - TCgTTCggggTACCgATgggg- 3, (SEQ ID NO: 84)
5， -TCgTTgCgCTCCCATgCCgggggg-3 ' (SEQ ID NO :85)  5, -TCgTTgCgCTCCCATgCCgggggg-3 ' (SEQ ID NO:85)
5， -TCgTCgTTTCgTCgTTgggg-3 ' (SEQ ID NO :86)  5, -TCgTCgTTTCgTCgTTgggg-3 ' (SEQ ID NO: 86)
5， -TCgTTgTCgTTTCgCTgCCggCggggg-3 ' (SEQ ID NO :87)  5, -TCgTTgTCgTTTCgCTgCCggCggggg-3 ' (SEQ ID NO:87)
5' -TgCTTgggTggCAgCTgCCAgggggg-3 ' (SEQ ID NO: 125)  5' -TgCTTgggTggCAgCTgCCAgggggg-3 ' (SEQ ID NO: 125)
5， -TgCTgCTTTgCTgCTTgggg-3 ' (SEQ ID NO :126)  5, -TgCTgCTTTgCTgCTTgggg-3 ' (SEQ ID NO: 126)
5， -AACgTTCgACgTCgAACggggggg-3 ' (SEQ ID NO :163)  5, -AACgTTCgACgTCgAACggggggg-3 ' (SEQ ID NO : 163)
5， -AACgACgACgTTggggg- 3， (SEQ ID NO: 167)  5, -AACgACgACgTTggggg- 3, (SEQ ID NO: 167)
(4) (TCG DJ CG (M)n， 其中乂1为八， T或 G; ¾为 A或 T; L和 Μ分别为 Α， Τ， C或 G; η为 0-6的整数； (4) (TCG DJ CG (M) n , where 乂1 is eight, T or G; 3⁄4 is A or T; L and Μ are Α, Τ, C or G, respectively; η is an integer from 0-6;
优选地， 式 （4) 中人工合成的单链脱氧核苷酸具有选自下列任一所示的序列- Preferably, the artificially synthesized single-stranded deoxynucleotide of formula (4) has a sequence selected from any one of the following -
5' - TCgTAACgTTgTTTTTAACgTT- 3， (SEQ ID NO :9) 5' - TCgTAACgTTgTTTTTAACgTT- 3, (SEQ ID NO: 9)
5' -TCgTCgTATACgACgATCgTT-3 ' (SEQ ID NO :10)  5' -TCgTCgTATACgACgATCgTT-3 ' (SEQ ID NO : 10)
5， -TCgTCgTTTgCgTTgTCgTT-3 ' (SEQ ID NO: 12)  5, -TCgTCgTTTgCgTTgTCgTT-3 ' (SEQ ID NO: 12)
5， - TCCTgTCgTTTTgTCgTT- 3， (SEQ ID NO :14)  5, - TCCTgTCgTTTTgTCgTT- 3, (SEQ ID NO: 14)
5' -TCgTCgTTgTCgTTCgCT-3' (SEQ ID NO :15)  5' -TCgTCgTTgTCgTTCgCT-3' (SEQ ID NO: 15)
5， -TCgTCgTTACCgATgACgTCgCCgT-3 ' (SEQ ID NO: 17) ， -TCgTCgTTTgCATCgATgCAgTCgTCgTT-3' (SEQ ID NO :20) ， -TCgCCTCgTCgCCTTCgAgC-3 ' (SEQ ID NO :30)5, -TCgTCgTTACCgATgACgTCgCCgT-3 ' (SEQ ID NO: 17) , -TCgTCgTTTgCATCgATgCAgTCgTCgTT-3' (SEQ ID NO: 20), -TCgCCTCgTCgCCTTCgAgC-3 ' (SEQ ID NO: 30)
, -TCgTgTgCgTgCCgTTggg-3' (SEQ ID NO :31) , -TCgTgTgCgTgCCgTTggg-3' (SEQ ID NO: 31)
， -TCgTCgAgggCgCCggTgAC-3' (SEQ ID NO :32) , -TCgTCgAgggCgCCggTgAC-3' (SEQ ID NO:32)
， -TCgTCgCCggTgggggTgTg-3， (SEQ ID NO :33) , -TCgTCgCCggTgggggTgTg-3, (SEQ ID NO :33)
， -TCgTCgTACgCAATTgTCTT-3， (SEQ ID NO :34) , -TCgTCgTACgCAATTgTCTT-3, (SEQ ID NO: 34)
， -TCgCCCACCggTgggggggg-3， (SEQ ID NO :35) , -TCgCCCACCggTgggggggg-3, (SEQ ID NO: 35)
， -TCgTCgCAgACCggTCTgggg-3， (SEQ ID NO :36) , -TCgTCgCAgACCggTCTgggg-3, (SEQ ID NO :36)
， -TCgTCgCggCCggCgCCCCC-3， (SEQ ID NO :39) , -TCgTCgCggCCggCgCCCCC-3, (SEQ ID NO: 39)
， -TCgTCgCggCCgCgAggggg-3 ' (SEQ ID NO :40) , -TCgTCgCggCCgCgAggggg-3 ' (SEQ ID NO :40)
， -TCgAggACAAgATTCTCgTgC-3， (SEQ ID NO :41) , -TCgAggACAAgATTCTCgTgC-3, (SEQ ID NO: 41)
， -TCgAggACAAgATTCTCgTgCAggCC-3 ' (SEQ ID NO :42) ， - TCgTgCAggCCAACgAggCCg- 3， (SEQ ID NO :43) , -TCgAggACAAgATTCTCgTgCAggCC-3 ' (SEQ ID NO:42) , - TCgTgCAggCCAACgAggCCg- 3, (SEQ ID NO :43)
， - TCgTTgCCgTCggCCC- 3， (SEQ ID NO :45) , - TCgTTgCCgTCggCCC- 3, (SEQ ID NO: 45)
， -TCggCACgCgACgTgCTggCCgTCgTTTCC-3' (SEQ ID NO :47) ， -TCgTTgCCgTCggCCCCCCCCC-3 ' (SEQ ID NO :54) , -TCggCACgCgACgTgCTggCCgTCgTTTCC-3' (SEQ ID NO: 47) , -TCgTTgCCgTCggCCCCCCCCC-3 ' (SEQ ID NO: 54)
， -TCgTTgCCgTCggCCCCCC-3 ' (SEQ ID NO :55) , -TCgTTgCCgTCggCCCCCC-3 ' (SEQ ID NO : 55)
， - TCgTTgCCgTCggCCCCC- 3， (SEQ ID NO :56) , - TCgTTgCCgTCggCCCCC- 3, (SEQ ID NO: 56)
， - TCgTTgCCgTCggCCCC- 3， (SEQ ID NO :57) , - TCgTTgCCgTCggCCCC- 3, (SEQ ID NO : 57)
， - TCgTTgCCgTCggCCCCCCC- 3， (SEQ ID NO :58) , - TCgTTgCCgTCggCCCCCCC- 3, (SEQ ID NO: 58)
， -TCgAggACAAgATTCTCgT-3 ' (SEQ ID NO :67) , -TCgAggACAAgATTCTCgT-3 ' (SEQ ID NO :67)
， -TCggCACgCgACgTgCTggCCgTCgTT-3 ' (SEQ ID NO :69) ， - TCgTCgCgCCgTCACgggggg- 3， (SEQ ID NO :70) , -TCggCACgCgACgTgCTggCCgTCgTT-3 ' (SEQ ID NO: 69) , - TCgTCgCgCCgTCACgggggg-3, (SEQ ID NO: 70)
， -TCgTgTgCgTgCCgTTggg- 3， (SEQ ID NO :71) , -TCgTgTgCgTgCCgTTggg- 3, (SEQ ID NO: 71)
， - TCgTCgCCgTTgggCggg- 3， (SEQ ID NO :72) , - TCgTCgCCgTTgggCggg- 3, (SEQ ID NO: 72)
， - TCgTCgACgTCgTTgggCggg- 3， (SEQ ID NO :73) , - TCgTCgACgTCgTTgggCggg- 3, (SEQ ID NO: 73)
， -TCgCAgTTgTCgTAACgTTgggCggg-3 ' (SEQ ID NO :74) ， -TCgTCgTTggTATgTT-3' (SEQ ID NO :77) , -TCgCAgTTgTCgTAACgTTgggCggg-3 ' (SEQ ID NO: 74) , -TCgTCgTTggTATgTT-3' (SEQ ID NO: 77)
， - TCgTCgTCgTCgTTgTCgTT- 3， (SEQ ID NO :78) , - TCgTCgTCgTCgTGgTGgTT- 3, (SEQ ID NO: 78)
， -TCgTCgTCgTCgTTgTCgTTgggg-3 ' (SEQ ID NO :79) 5' -TCgTTCggggTgCCg-3' (SEQ ID NO :80) , -TCgTCgTCgTCgTTgTCgTTgggg-3 ' (SEQ ID NO :79) 5'-TCgTTCggggTgCCg-3' (SEQ ID NO: 80)
5， -TCgTTCggggTAACgATT-3 ' (SEQ ID NO :81)  5, -TCgTTCggggTAACgATT-3 ' (SEQ ID NO :81)
5， - TCgTTCggggTAACgTT- 3， (SEQ ID NO :82)  5, - TCgTTCggggTAACgTT- 3, (SEQ ID NO: 82)
5， -TCgTTCggggTACCgAT- 3， (SEQ ID NO :83)  5, -TCgTTCggggTACCgAT- 3, (SEQ ID NO: 83)
5， -TCgTACggCCgCCgTACggCggg-3 ' (SEQ ID NO :92)  5, -TCgTACggCCgCCgTACggCggg-3 ' (SEQ ID NO: 92)
5， -TCgCgTCgACTCCCCTCgAgggg-3 ' (SEQ ID NO :94)  5, -TCgCgTCgCCCCCCTCgAgggg-3 ' (SEQ ID NO: 94)
5， -TCgTCgTCgACTCgTggTCggggg-3 ' (SEQ ID NO :95)  5, -TCgTCgTCgACTCgTggTCggggg-3 ' (SEQ ID NO: 95)
5， -TCgggCgCCCgATCgggggg-3 ' (SEQ ID NO :96)  5, -TCgggCgCCCgATCgggggg-3 ' (SEQ ID NO : 96)
5，—TCgTCggTCTTTCgAAATT— 3， (SEQ ID NO :97)  5, —TCgTCggTCTTTCgAAATT— 3, (SEQ ID NO: 97)
5， - TCgTgACgTCCTCgAgTT- 3， (SEQ ID NO :98)  5, - TCgTgACgTCCTCgAgTT-3, (SEQ ID NO: 98)
5， -TCgTCTTTCgACTCgTTCTC-3 ' (SEQ ID NO: 115)  5, -TCgTCTTTCgACTCgTTCTC-3 ' (SEQ ID NO: 115)
5， - TCgTCgTTTTgCgTTCTC-3' (SEQ ID NO :116)  5, - TCgTCgTTTTgCgTTCTC-3' (SEQ ID NO: 116)
5， - TCgACTTTCgTCgTTCTgTT - 3， (SEQ ID NO: 119)  5, - TCgACTTTCgTCgTTCTgTT - 3, (SEQ ID NO: 119)
5， - TCgTCgTTTCgTCgTTCTC - 3， (SEQ ID NO :120)  5, - TCgTCgTTTCgTCgTTCTC - 3, (SEQ ID NO : 120)
5， -TCgTCgTCgTCgTTgTCgTT-3 ' (SEQ ID NO: 121)  5, -TCgTCgTCgTCgTGgTCgTT-3 ' (SEQ ID NO: 121)
5， -TCgTTCTCgACTCgTTCTC-3 ' (SEQ ID NO: 122)  5, -TCgTTCTCgACTCgTTCTC-3 ' (SEQ ID NO: 122)
5， - TCgACgTTCgTCgTTCgTCgTTC- 3， (SEQ ID NO :123)  5, - TCgACgTTCgTCgTTCgTCgTTC- 3, (SEQ ID NO: 123)
5， -TCgTCgACgTCgTTCgTTCTC-3 ' (SEQ ID NO: 124)  5, -TCgTCgACgTCgTTCgTTCTC-3 ' (SEQ ID NO: 124)
5， -TCgTgCgACgTCgCAgATgAT-3 ' (SEQ ID NO: 138)  5, -TCgTgCgACgTCgCAgATgAT-3 ' (SEQ ID NO: 138)
5， -TCgTCgAgCgCTCgATCggAT-3 ' (SEQ ID NO :141)  5, -TCgTCgAgCgCTCgATCggAT-3 ' (SEQ ID NO: 141)
5' -TCgTCgTTTCgTAgTCgTTgACgTCggg-3' (SEQ ID NO :142)  5' -TCgTCgTTTCgTAgTCgTTgACgTCggg-3' (SEQ ID NO: 142)
5， -TCgTCggACgTTTTCCgACgTTCT-3 ' (SEQ ID NO :144)  5, -TCgTCggACgTTTTCCgACgTTCT-3 ' (SEQ ID NO: 144)
5， - TCgTCgTTTTCgTCgTTTTCgTCgTT- 3， (SEQ ID NO: 151)  5, - TCgTCgTTTTCgTCgTTTTCgTCgTT- 3, (SEQ ID NO: 151)
5， -TCgTCgTTTgTCgTgTgTCgTT-3 ' (SEQ ID NO: 152)  5, -TCgTCgTTTgTCgTgTgTCgTT-3 ' (SEQ ID NO: 152)
5， -TCgTCgTTggTCggggTCgTTggggTCgTT-3' (SEQ ID NO: 153)  5, -TCgTCgTTggTCggggTCgTTggggTCgTT-3' (SEQ ID NO: 153)
5， -TCgTCgTTTCgTCTCTCgTT-3 ' (SEQ ID NO :154)  5, -TCgTCgTTTCgTCTCTCgTT-3 ' (SEQ ID NO: 154)
5' -TCgTCgTTTTgCTgCgTCgTT-3 ' (SEQ ID NO: 155)  5' -TCgTCgTTTTgCTgCgTCgTT-3 ' (SEQ ID NO: 155)
5， - TCgAgCgTTTTCgCTCgAATT— 3， (SEQ ID NO :178)  5, - TCgAgCgTTTTCgCTCgAATT-3, (SEQ ID NO: 178)
(5)包含 TTCGTCG的序列。 (5) A sequence containing TTCGTCG.
优选地， 式 （5) 中人工合成的单链脱氧核苷酸具有选自下列任一所示的序列: 5' - TTCgTCgTTTgATCgATgTTCgTTgggggg- 3， (SEQ ID NO :25) 'Preferably, the artificially synthesized single-stranded deoxynucleotide of formula (5) has a sequence selected from any one of the following: 5' - TTCgTCgTTTgATCgATgTTCgTTgggggg- 3, (SEQ ID NO: 25) '
5， -TTCgTCgTTgTgATCgATgggggg-3 ' (SEQ ID NO : 26) 5, -TTCgTCgTTgTgATCgATgggggg-3 ' (SEQ ID NO : 26)
5， -TATCgATgTTTTCgTCgTCgTTgggggg-3' (SEQ ID NO : 27)  5, -TATCgATgTTTTCgTCgTCgTTgggggg-3' (SEQ ID NO: 27)
5， -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 119)  5, -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 119)
5， -TCgTCgTTTCgTCgTTCTC-3 ' (SEQ ID NO : 120)  5, -TCgTCgTTTCgTCgTTCTC-3 ' (SEQ ID NO : 120)
5， -TCgACgTTCgTCgTTCgTCgTTC- 3， (SEQ ID NO : 123)  5, -TCgACgTTCgTCgTTCgTCgTTC- 3, (SEQ ID NO: 123)
5' -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 151)  5' -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 151)
本领域的普通技术人员可以了解， 包含一个或多个上式序列的人工合成的单链脱 氧核苷酸均可以用于实现本发明的目的。  One of ordinary skill in the art will appreciate that synthetic single-stranded deoxynucleotides comprising one or more of the above sequences can be used to accomplish the objectives of the present invention.
本发明所述的人工合成的单链脱氧核苷酸还包括单链脱氧核苷酸中碱基上各个 基团的修饰， 其中所述的修饰包括非硫代修饰、 硫代修饰、 部分硫代修饰、 稀有碱基 修饰 （dl， dU等）、 甲基化修饰、 巯基、 Aminol inker C6s 或 Thiol- C6 S- S等用于与 其他物质偶联所应用的修饰方式。 上述修饰对于本领域的普通技术人员而言是熟知 的。  The artificially synthesized single-stranded deoxynucleotide of the present invention further comprises a modification of each group in the base of the single-stranded deoxynucleotide, wherein the modification includes a non-thio modification, a thio modification, a partial thio Modifications, rare base modifications (dl, dU, etc.), methylation modifications, thiol groups, Aminol inker C6s or Thiol-C6 S-S, etc., are used in conjunction with other materials. Such modifications are well known to those of ordinary skill in the art.
本发明的另一个目的是提供由上述单链脱氧核苷酸与抗原或抗原组合物所组成 的疫苗组合物， 其中抗原或抗原组合物的优选为由分枝杆菌热休克蛋白 65和抗原肽形 成的复合物或融合蛋白， 该复合物或融合蛋白为能诱生抗原特异性 CTL的制剂。 所述 的抗原肽是指具有免疫原性的肽段或在和其它的蛋白质形成复合物、 偶连物或融合蛋 白状态下具有免疫原性的肽段， 例如： 匿 122116. 2 中的多表位丙型肝炎病毒核心 抗原抗原肽， 该抗原肽对丙型肝炎病毒感染和丙型肝炎病毒感染引起的相关疾病具有 预防和治疗作用， 具体序列如下：  Another object of the present invention is to provide a vaccine composition comprising the above-described single-stranded deoxynucleotide and an antigen or antigen composition, wherein the antigen or antigen composition is preferably formed of mycobacterial heat shock protein 65 and an antigen peptide A complex or fusion protein that is a preparation that induces antigen-specific CTL. The antigenic peptide refers to a peptide segment having immunogenicity or a peptide having immunogenicity in a state of forming a complex, a conjugate or a fusion protein with other proteins, for example: a multi-table in 122116. Hepatitis C virus core antigen antigen peptide, which has preventive and therapeutic effects on diseases caused by hepatitis C virus infection and hepatitis C virus infection, and the specific sequence is as follows:
Met Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys Glu lie Asp  Met Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys Glu lie Asp
Asp Phe Ala Asp Leu Met Gly Tyr lie Pro Leu Val Gly Ala Pro  Asp Phe Ala Asp Leu Met Gly Tyr lie Pro Leu Val Gly Ala Pro
Leu Glu Asp Ser Glu Gly Val Tyr Leu Leu Pro Arg Arg Gly 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 lie Glu Gly Pro Arg  Arg Leu Gly Val Arg Ala Glu Asn Asp Glu lie Glu Gly Pro Arg
Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Asn Asp Glu  Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Asn Asp Glu
Leu Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin  Leu Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin
Glu lie Asp Asn Glu (SEQ ID NO: 181 )  Glu lie Asp Asn Glu (SEQ ID NO: 181 )
CN02141977. 9中的沙眼衣原体主要为多表位抗原多肽， 其对由衣原体和衣原体 感染引起的相关疾病具有治疗和预防的作用， 具体序列如下： Glu Phe Pro Ala Tyr Gly Arg His Met Gin Asp Ala Glu Met Phe Thr Asn Ala Ala Cys Met Ala Leu Asn lie Trp Asp Glu Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr lie 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 lie Asp Tyr His Glu Trp Gin Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr lie Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp Thr Tyr Lys Leu (SEQ ID NO : 182) CN02141977. The Chlamydia trachomatis in 9 is mainly a multi-epitope antigen polypeptide, which has therapeutic and preventive effects on related diseases caused by infection of Chlamydia and Chlamydia, and the specific sequences are as follows: Glu Phe Pro Ala Tyr Gly Arg His Met Gin Asp Ala Glu Met Phe Thr Asn Ala Ala Cys Met Ala Leu Asn lie Trp Asp Glu Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr lie 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 lie Asp Tyr His Glu Trp Gin Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr lie Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp Thr Tyr Lys Leu (SEQ ID NO : 182)
CN01134935. 2中的人前列腺特异性抗原细胞毒性 T淋巴细胞多表位的单拷贝抗原 多肽， 其对人的前列腺癌具有预防和治疗的作用， 具体序列如下：  CN01134935. Human prostate specific antigen cytotoxicity T lymphocyte multi-epitope single copy antigen polypeptide, which has preventive and therapeutic effects on human prostate cancer, and the specific sequence is as follows:
Phe Leu Thr Pro Lys Lys Leu Gin Cys Val Asp Leu His Val lie Ser Phe Leu Thr Pro Lys Lys Leu Gin Cys Val Asp Leu His Val lie Ser
Asn Asp Val Cys Ala Gin Val His Pro Gin Lys Val Thr Lys (SEQ ID NO : 183)Asn Asp Val Cys Ala Gin Val His Pro Gin Lys Val Thr Lys (SEQ ID NO : 183)
CN011026146中的 MUC1抗原细胞毒性 T淋巴细胞表位抗原肽， 其对表达 MUC1的肿 瘤包括但不限于乳腺癌、 卵巢癌、 肺癌、 前列腺癌、 结直肠癌细胞等具有预防和治疗 的作用， 具体序列如下： The MUC1 antigen cytotoxic T lymphocyte epitope antigen peptide in CN011026146, which has preventive and therapeutic effects on tumors expressing MUC1 including, but not limited to, breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer cells, etc., specific sequence as follows:
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp  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 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu (SEQ ID NO : 184)  Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu (SEQ ID NO : 184)
CN011363479中的多表位 HER- 2抗原肽， 其对表达 HER-2的肿瘤包括但不限于乳 腺癌、 卵巢癌、 胃癌、 子宫内膜癌、 唾液腺癌、 腺癌、 前腺癌、 结肠和直肠癌、 非小 细胞肺癌、 肺腺癌等具有治疗和预防的作用， 具体序列如下- The multi-epitope HER-2 antigen peptide of CN011363479 for tumors expressing HER-2 including, but not limited to, breast cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, adenocarcinoma, anterior adenocarcinoma, colon and rectum Cancer, non-small cell lung cancer, lung adenocarcinoma, etc. have therapeutic and preventive effects, the specific sequence is as follows -
Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala Asn lie Gin Glu Phe Ala Gly Cys Lys Lys lie Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gin Pro Glu Gin Leu Gin Val Phe Glu Thr Leu Glu Glu lie (SEQ ID NO : 185) 但本领域的普通技术人员可以理解， 用于本发明的抗原或抗原组合物的抗原肽 并不限于上述实例。 · Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala Asn lie Gin Glu Phe Ala Gly Cys Lys Lys lie Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gin Pro Glu Gin Leu Gin Val Phe Glu Thr Leu Glu Glu lie (SEQ ID NO: 185) However, it will be understood by one of ordinary skill in the art that the antigenic peptide used in the antigen or antigen composition of the present invention is not limited to the above examples. ·
本发明的另一目的在于提供一种生产疫苗组合物的方法， 包括将有效量的上述人 工合成的单链脱氧核苷酸与有效量的抗原或抗原组合物混合。 优选将有效量的人工合 成的单链脱氧核苷酸与有效量的由分枝杆菌热休克蛋白 65和抗原肽形成的复合物或 融合蛋白相混合'。 本发明的另一方面提供了一种增强抗原或抗原组合物免疫剌激作用的方法， 其是 将人工合成的单链脱氧核苷酸与抗原或抗原组合物联合使用。 Another object of the present invention is to provide a method of producing a vaccine composition comprising mixing an effective amount of the above-described synthetic single-stranded deoxynucleotide with an effective amount of an antigen or antigen composition. Preferably, an effective amount of the synthetic single-stranded deoxynucleotide is mixed with an effective amount of a complex or fusion protein formed by the mycobacterial heat shock protein 65 and the antigenic peptide. Another aspect of the invention provides a method of enhancing the immunostimulatory effect of an antigen or antigen composition by using a synthetic single-stranded deoxynucleotide in combination with an antigen or antigen composition.
本发明的研究结果表明， 当人工合成的单链脱氧核苷酸与由分枝杆菌热休克蛋白 The results of the present invention indicate that when the synthetic single-stranded deoxynucleotides are combined with heat shock proteins by mycobacteria
65和抗原肽形成的复合物或融合蛋白混合使用时， 人工合成的单链脱氧核苷酸能够显 著增强分枝杆菌热休克蛋白 65抗原肽融合蛋白诱生抗原肽特异性 CTL的活性， 显著增 强分枝杆菌热休克蛋白 65抗原肽融合蛋白刺激小鼠抑制表达抗原肽肿瘤细胞生长的 活性， 显著增强分枝杆菌热休克蛋白 65抗原肽融合蛋白诱生抗原肽特异性的抗肿瘤、 抗病毒和抗衣原体感染的活性。 When a complex or fusion protein formed with an antigen peptide is used in combination, the synthetic single-stranded deoxynucleotide can significantly enhance the activity of the mycobacterial heat shock protein 65 antigen peptide fusion protein to induce antigenic peptide-specific CTL, which is significantly enhanced. Mycobacterial heat shock protein 65 antigen peptide fusion protein stimulates mouse to inhibit the growth of antigenic peptide-expressing tumor cells, and significantly enhances the anti-tumor, anti-virus and anti-tumor specificity of mycobacterial heat shock protein 65 antigen peptide fusion protein-induced antigen peptide Anti-Chlamydia infection activity.
本发明的再一方面是提供疫苗组合物在制备用于治疗病毒感染、 细菌感染、 寄 生虫感染、 变态反应或癌症的疫苗中的应用。 包括将有效量的由人工合成的单链脱氧 核苷酸与抗原或抗原组合物组成的免疫组合物施与需要治疗的人或哺乳动物。 所述的 病毒感染、 细菌感染、 寄生虫感染、 变态反应或癌症包括但并不局限于由丙型肝炎病 毒感染引起的相关疾病、 由衣原体和衣原体感染引起的相关疾病、前列腺癌、乳腺癌、 卵巢癌、 肺癌、 胃癌、 子宫内膜癌、 唾液腺癌、 腺癌、 结肠和直肠癌、 非小细胞肺癌、 肺腺癌等。 具体实施方式  A further aspect of the invention is the use of a vaccine composition for the preparation of a vaccine for the treatment of a viral infection, a bacterial infection, a parasitic infection, an allergy or cancer. This includes administering an effective amount of an immunological composition consisting of a synthetic single-stranded deoxynucleotide to an antigen or antigen composition to a human or mammal in need of treatment. The viral infection, bacterial infection, parasitic infection, allergy or cancer includes, but is not limited to, related diseases caused by hepatitis C virus infection, related diseases caused by infection of chlamydia and chlamydia, prostate cancer, breast cancer, Ovarian cancer, lung cancer, gastric cancer, endometrial cancer, salivary gland cancer, adenocarcinoma, colon and rectal cancer, non-small cell lung cancer, lung adenocarcinoma, and the like. detailed description
下面结合具体的制备实施例和生物学效果实施例， 进一步详细地描述本发明。 本 领域的普通技术人员应理解， 这些实施例只是为了举例说明本发明， 而非以任何方式 限制本发明的范围。  The invention is described in further detail below in conjunction with specific preparation examples and biological effect examples. It is to be understood by those skilled in the art that these embodiments are only intended to illustrate the invention and not to limit the scope of the invention in any way.
在如下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法， 例 如合成采用固相亚磷酰胺三酯法、 ELBA采用间接法。  In the following examples, various processes and methods not described in detail are conventional methods well known in the art, such as the solid phase phosphoramidite triester method and the ELBA indirect method.
在如下实施例中， 所用试剂的来源、 商品名和 /或有必要列出其组成成分者， 均 只标明一次。 在其后所用相同试剂如无特殊说明， 不在赘述上述内容。 实施例 1 人工合成单链脱氧核苷酸的设计  In the following examples, the source of the reagents used, the trade name, and/or the components that are necessary to list them are indicated only once. The same reagents used hereinafter are not described above unless otherwise stated. Example 1 Design of synthetic single-stranded deoxynucleotides
设计序列如下- ( 1 ) (G)n(L)n X1X2CGY1Y2(M)n (G)n The design sequence is as follows - ( 1 ) (G) n (L) n X 1 X 2 CGY 1 Y 2 (M) n (G) n
¾=A, T或 G; X2=A或 Τ; Υ尸 Α或 Τ; Υ2=Α, Τ或 C; L， M=A, T， C或 G; n为 0-6 3⁄4=A, T or G; X 2 = A or Τ; Υ Α or Τ; Υ 2 = Α, Τ or C; L, M=A, T, C or G; n is 0-6
5， -ggggTCgTTCgTCgTTgggggg-3 ' (SEQ ID NO : 2) Ss00/:l>d 16S.0/ O900ZAV ) (，¾vvvNs〕l¾ O GI 53sJ寸ssssgs：sss—l 5, -ggggTCgTTCgTCgTTgggggg-3 ' (SEQ ID NO : 2) Ss00/:l>d 16S.0/ O900ZAV ) (,3⁄4vvvNs]l3⁄4 O GI 53sJ inch ssssgs:sss-l
()，¾vx£lv¾〕8ε ON ai &HS>ssss:ss§.-- (), 3⁄4vx£lv3⁄4]8ε ON ai &HS>ssss:ss§.--
Figure imgf000015_0001
β ( ,vvi9 i ON aiT¾v¾9sSS:SSS—
Figure imgf000015_0001
β ( ,vvi9 i ON aiT3⁄4v3⁄49sSS:SSS—
LO ) (，Ι 5¾v9ΟΝαssv¾310ΐ3s:sssssss—- § (vvv¾〕l¾v¾〕0Ν αιΟs:sssss--  LO ) (,Ι 53⁄4v9ΟΝαssv3⁄4310ΐ3s:sssssss—- § (vvv3⁄4]l3⁄4v3⁄4]0Ν αιΟs:sssss--
) (¾vI gs¾¾lv0Ν Gv38ΐ¾l0:§ssss§-- ) βs¾〕 αι,l〕ΟΝJ¾ggsss:gs-- § ( αι3 ON¾¾9,ssss:§sss-l 5' -TTCgTTgCATCgATgCATCgTTgggggg-3' (SEQ ID NO :28) ) (3⁄4vI gs3⁄43⁄4lv0Ν Gv38ΐ3⁄4l0:§ssss§-- ) βs3⁄4] αι,l〕ΟΝJ3⁄4ggsss:gs-- § ( αι3 ON3⁄43⁄49,ssss:§sss-l 5'-TTCgTTgCATCgATgCATCgTTgggggg-3' (SEQ ID NO: 28)
(2) (G)n(L)nCG(XY)nCG(M)n(G)n (2) (G)n(L) n CG(XY) n CG(M)n(G)n
X=A或 T; Y=A或 T; L， M=A， T， C或 G; n为 0-6 X=A or T; Y=A or T; L, M=A, T, C or G; n is 0-6
5' -ggggACgATACgTCggggggg-3 ' (SEQ ID N0:1) 5' -ggggACgATACgTCggggggg-3 ' (SEQ ID N0:1)
5' -ggggACgATATCgATgggggg-3 ' (SEQ ID NO :7) 5' -ggggACgATATCgATgggggg-3 ' (SEQ ID NO :7)
- ggACgATCgATCgTgggggg-3' (SEQ ID NO :99)  - ggACgATCgATCgTgggggg-3' (SEQ ID NO: 99)
-TCggggACgATCgTCgggggg-3' (SEQ ID NO :106)  -TCggggACgATCgTCgggggg-3' (SEQ ID NO: 106)
-gggggATCgATATCgATCgggggg-3' (SEQ ID NO :107) ggATCgATCgATCgATgggggg- 3' (SEQ ID NO: 110)  -gggggATCgATATCgATCgggggg-3' (SEQ ID NO:107) ggATCgATCgATCgATgggggg-3' (SEQ ID NO: 110)
-ggTgCATCgATCgATgCAgggggg-3' (SEQ ID NO :109) '- ggTgCATCgTACgATgCAgggggg- 3' (SEQ ID NO: 104) -ggTgCgATCgATCgCAgggggg-3' (SEQ ID NO: 113) -gggggggTCgATCgATgggggg-3 ' (SEQ ID NO: 171) -ggggTCgTCgAACgTTgggggg-3 ' (SEQ ID NO :172) - TgTCgTTCCTTgTCgTT- 3， (SEQ ID NO :16)  -ggTgCATCgATCgATgCAgggggg-3' (SEQ ID NO: 109) '- ggTgCATCgTACgATgCAgggggg-3' (SEQ ID NO: 104) -ggTgCgATCgATCgCAgggggg-3' (SEQ ID NO: 113) -gggggggTCgATCgATgggggg-3 ' (SEQ ID NO: 171) - ggggTCgTCgAACgTTgggggg-3 ' (SEQ ID NO: 172) - TgTCgTTCCTTgTCgTT-3, (SEQ ID NO: 16)
-TTCgCTTCgCTTTTCgCTTCgCTT-3 ' (SEQ ID NO :29) -ACCgCCAAggAgAAgCCgCAggAggg-3 ' (SEQ ID NO :44) - TACAACggCgAggAATACC- 3， (SEQ ID NO :46)  -TTCgCTTCgCTTTTCgCTTCgCTT-3 ' (SEQ ID NO: 29) - ACCgCCAAggAgAAgCCgCAggAggg-3 ' (SEQ ID NO: 44) - TACAACggCgAggAATACC-3, (SEQ ID NO: 46)
-gTACAACggCgAggAATACCT-3 ' (SEQ ID NO :48)  -gTACAACggCgAggAATACCT-3 ' (SEQ ID NO :48)
-ACCgTCgTTgCCgTCggCCC-3 ' (SEQ ID NO :49)  -ACCgTCgTGgCCgTCggCCC-3 ' (SEQ ID NO: 49)
-TgCTggCCgTCgTT- 3， (SEQ ID NO :50)  -TgCTggCCgTCgTT- 3, (SEQ ID NO: 50)
-gTCggCACgCgACg-3' (SEQ ID NO :51)  -gTCggCACgCgACg-3' (SEQ ID NO: 51)
-gTCggCACgCgACgCCCCCC-3 ' (SEQ ID NO :53)  -gTCggCACgCgACgCCCCCC-3 ' (SEQ ID NO: 53)
-TCCCgCTggACgTT-3' (SEQ ID NO :68)  -TCCCgCTggACgTT-3' (SEQ ID NO: 68)
-TTACCggTTAACgTTggCCggCC-3 ' (SEQ ID NO :75) -ACCggTTAACgTTgTCCCCgggg-3 ' (SEQ ID NO :76) -CgTTgACgATCgTCCCATggCggg-3 ' (SEQ ID NO :88) -TCTgCggCCTTCgTCg-3' (SEQ ID NO :89)  -TTACCggTTAACgTTggCCggCC-3 ' (SEQ ID NO:75) -ACCggTTAACgTTgTCCCCgggg-3 ' (SEQ ID NO:76) -CgTTgACgATCgTCCCATggCggg-3 ' (SEQ ID NO:88) -TCTgCggCCTTCgTCg-3' (SEQ ID NO:89)
-TAgTAACCggTCCggCgCCCCC-3 ' (SEQ ID NO :90) -TTgCAgCgCTgCCggTggg-3' (SEQ ID NO :91) ， -CggCCCATCgAgggCgACggC-3 ' (SEQ ID NO :93) ， -TCATCgACTCTCgAgCgTTC-3 ' (SEQ ID NO: 117) ， -ATCgTCgACTCTCgTgTTCTC-3 ' (SEQ ID NO :118) ， -TgCAgCTTgCTgCTTgCTgCTTC-3 ' (SEQ ID NO: 127) ， -ggTgCgACgTCgCAgATgAT-3 ' (SEQ ID NO: 136) ， -ggTCgAACgTTCgAgATgAT-3 ' (SEQ ID NO :137) ， -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 143) ， -actcgagacgcccgttgatagctt-3 ' (SEQ ID NO: 145) ， -AACgTTggCgTCgACgTCAgCgCC-3 ' (SEQ ID NO: 146) ， -gACgTCgACgTTgACgCT-3 ' (SEQ ID NO: 147)-TAgTAACCggTCCggCgCCCCC-3 ' (SEQ ID NO: 90) -TTgCAgCgCTgCCggTggg-3' (SEQ ID NO: 91) , -CggCCCATCgAgggCgACggC-3 ' (SEQ ID NO:93) , -TCATCgACTCTCgAgCgTTC-3 ' (SEQ ID NO: 117) , -ATCgTCgACTCTCgTgTTCTC-3 ' (SEQ ID NO:118) , -TgCAgCTTgCTgCTTgCTgCTTC-3 ' (SEQ ID NO: 127), -ggTgCgACgTCgCAgATgAT-3 ' (SEQ ID NO: 136), -ggTCgAACgTTCgAgATgAT-3 ' (SEQ ID NO: 137), -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 143) , -actcgagacgcccgttgatagctt-3 ' (SEQ ID NO: 145) , -AACgTTggCgTCgACgTCAgCgCC-3 ' (SEQ ID NO: 146) , -gACgTCgACgTTgACgCT-3 ' (SEQ ID NO: 147)
, -ggCgTTAACgTTAgCgCT-3' (SEQ ID NO :148)  , -ggCgTTAACgTTAgCgCT-3' (SEQ ID NO: 148)
， -AgCgCTAgCgCTgACgTT-3 ' (SEQ ID NO :149)  , -AgCgCTAgCgCTgACgTT-3 ' (SEQ ID NO :149)
， - CTAgACgTTCAAgCgTT- 3， (SEQ ID NO :150)  , - CTAgACgTTCAAgCgTT-3, (SEQ ID NO: 150)
， - gACgATCgTCgACgATCgTC- 3， (SEQ ID NO: 156) ' -gTCgTTCgTAgTCgACTACgAgTT-3 ' (SEQ ID NO: 161) ， -AAAAgACgTCgACgTCgACgTCTTTT-3 ' (SEQ ID NO: 162) ， -TgCgACgATCgTCgCACgATCggAT-3 ' (SEQ ID NO: 176) , - gACgATCgTCgACgATCgTC-3, (SEQ ID NO: 156) '-gTCgTTCgTAgTCgACTACgAgTT-3 ' (SEQ ID NO: 161), -AAAAgACgTCgACgTCgACgTCTTTT-3 ' (SEQ ID NO: 162) , -TgCgACgATCgTCgCACgATCggAT-3 ' (SEQ ID NO: 176)
5' -TgCgACgTCgCACAgCgT-3 ' (SEQ ID NO: 177) 5' -TgCgACgTCgCACAgCgT-3 ' (SEQ ID NO: 177)
(3) (TCG)n(L)nCG(M)n(G)n (3) (TCG) n (L) n CG(M) n (G) n
L， M=A， T， C或 G; n为 0-6  L, M=A, T, C or G; n is 0-6
， -TCgTTgCCgTCgg-3' (SEQ ID NO :59) , -TCgTTgCCgTCgg-3' (SEQ ID NO: 59)
' - TCgTTgCCgTCggg- 3， (SEQ ID NO :60)  ' - TCgTTgCCgTCggg- 3, (SEQ ID NO : 60)
， -TCgTTgCCgTCgggg-3' (SEQ ID NO :61)  , -TCgTTgCCgTCgggg-3' (SEQ ID NO: 61)
， - TCgTTgCCgTCggggg- 3， (SEQ ID NO :62) , - TCgTTgCCgTCggggg- 3, (SEQ ID NO : 62)
' -TCgTTgCCgTCgggggg-3' (SEQ ID NO :63)  ' -TCgTTgCCgTCgggggg-3' (SEQ ID NO: 63)
， -TCgTTgCCgTCggggggg-3' (SEQ ID NO :64)  , -TCgTTgCCgTCggggggg-3' (SEQ ID NO: 64)
， -TCgTTgCCgTCgggggggg-3' (SEQ ID NO :65) , -TCgTTgCCgTCgggggggg-3' (SEQ ID NO: 65)
' -TCgTTgCCgTCggggggggg-3 ' (SEQ ID NO :66) ' -TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO :103) ， -TCgTCgggTgCAACgTTgCAgggggg-3 ' (SEQ ID NO :129) ， -TCgTCgggTgCgTCgACgCAgggggg-3 ' (SEQ ID NO: 130) ， -TCgTCgggTgCgATCgCAgggggg-3 ' (SEQ ID NO : 131) ， -TCgTCgggTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO : 132) ， -TCgTCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 133) ， -TCgTCgCAgAACgTTCTgggggg-3 ' (SEQ ID NO : 134) '-TCgTTgCCgTCggggggggg-3 ' (SEQ ID NO: 66) '-TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO:103) , -TCgTCgggTgCAACgTTgCAgggggg-3 ' (SEQ ID NO:129) , -TCgTCgggTgCgTCgACgCAgggggg-3 ' (SEQ ID NO: 130), -TCgTCgggTgCgATCgCAgggggg-3 ' (SEQ ID NO: 131), -TCgTCgggTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO: 132), -TCgTCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 133) , -TCgTCgCAgAACgTTCTgggggg-3 ' (SEQ ID NO : 134)
， -TCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 135) , -TCgTgCgACgTCgCAgggggg-3 ' (SEQ ID NO : 135)
' -TCgTgCgACgATCgTCgCAgggggg-3 ' (SEQ ID NO : 139) ， - TCgTATgCATCgATgCATAgggAgg- 3， (SEQ ID NO : 140) ' - TCgTgCATCgATgCAgggggg- 3， (SEQ ID NO : 157)  '-TCgTgCgACgATCgTCgCAgggggg-3 ' (SEQ ID NO: 139), - TCgTATgCATCgATgCATAgggAgg-3, (SEQ ID NO: 140) ' - TCgTgCATCgATgCAgggggg-3, (SEQ ID NO: 157)
， -TCgMACgTTTCgggggg- 3， (SEQ ID NO: 158)  , -TCgMACgTTTCgggggg- 3, (SEQ ID NO: 158)
， -TCggACgATCgTCgggggg-3' (SEQ ID NO : 159)  , -TCggACgATCgTCgggggg-3' (SEQ ID NO : 159)
， -TCgAgCgATCgCTCgAgggggg-3 ' (SEQ ID NO : 179)  , -TCgAgCgATCgCTCgAgggggg-3 ' (SEQ ID NO : 179)
， -TCgTCgCTTTgTCgTTgggg-3 ' (SEQ ID NO : 13)  , -TCgTCgCTTTgTCgTTgggg-3 ' (SEQ ID NO : 13)
， -TCgTCgTTTTgTCgTTgggg-3 ' (SEQ ID NO : 11) , -TCgTCgTTTTgTCgTTgggg-3 ' (SEQ ID NO : 11)
' -TCgTCgggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 18) ， -TCgTCgggTgCgACgATCgTCgggggg-3 ' (SEQ ID NO: 19) ， -TCgTCgTTTgCATCgATgCAggggggg-3 ' (SEQ ID NO: 21) ' -TCgTCgTTTTgACgATCgTCgggggg-3 ' (SEQ ID NO :24) ， -TCgTTCggggTgCCg-3' (SEQ ID NO : 80)  '-TCgTCgggTgCgACgTCgCAgggggg-3 ' (SEQ ID NO: 18), -TCgTCgggTgCgACgATCgTCgggggg-3 ' (SEQ ID NO: 19), -TCgTCgTTTgCATCgATgCAggggggg-3 ' (SEQ ID NO: 21) ' -TCgTCgTTTTgACgATCgTCgggggg-3 ' (SEQ ID NO : 24) , -TCgTTCggggTgCCg-3' (SEQ ID NO : 80)
， -TCgTTCggggTACCgATgggg-3 ' (SEQ ID NO :84)  , -TCgTTCggggTACCgATgggg-3 ' (SEQ ID NO :84)
， -TCgTTgCgCTCCCATgCCgggggg-3 ' (SEQ ID NO : 85)  , -TCgTTgCgCTCCCATgCCgggggg-3 ' (SEQ ID NO : 85)
， - TCgTCgTTTCgTCgTTgggg- 3，. (SEQ ID NO : 86)  , - TCgTCgTTTCgTCgTTgggg- 3,. (SEQ ID NO : 86)
， -TCgTTgTCgTTTCgCTgCCggCggggg-3 ' (SEQ ID NO : 87) ， -TgCTTgggTggCAgCTgCCAgggggg-3 ' (SEQ ID NO: 125) ， - TgCTgCTTTgCTgCTTgggg- 3， (SEQ ID NO : 126)  , -TCgTTgTCgTTTCgCTgCCggCggggg-3 ' (SEQ ID NO: 87) , -TgCTTgggTggCAgCTgCCAgggggg-3 ' (SEQ ID NO: 125) , - TgCTgCTTTgCTgCTTgggg-3, (SEQ ID NO: 126)
， -AACgTTCgACgTCgAACggggggg-3 ' (SEQ ID NO: 163) , -AACgTTCgACgTCgAACggggggg-3 ' (SEQ ID NO: 163)
5， -AACgACgACgTTggggg-3 ' (SEQ ID NO: 167) 5, -AACgACgACgTTggggg-3 ' (SEQ ID NO: 167)
(4) (TCG)n(L)nX1X2CG (M)„(4) (TCG) n (L) n X 1 X 2 CG (M) „
Figure imgf000018_0001
T， C或 G; n为 0-6; ， -TCgTAACgTTgTTTTTAACgTT-3 ' (SEQ ID NO : 9) ggggggggg)Q ( :72TccTTc3S ID NOccET- - ggggggggg) (Q1cEO :7cTcTTS I NTcTDT - ggggggg)ggg (Q0 NO :7cCATSE IDcTcccTClT— - )gggggggggg (QD NO :69S IcTEccAccAcTcTcTcT3T- - ggggg) (QO :67TTCTCT3SE ID NcAACAAATI - g)gggg (Q058ccccSED N:cTcTcccc3 ITcT— - ggggg) (QO57ccccc3SE I N :TcTDcTcT- -- gggggs (Q"cccc3SE ID §。ccTccTTT— - ggggsg (Qocc3SE I z"cTcccccDcTTcT— - ggg)gg (QO :5cc ID N4cccccccc3SEcTTccTT- - gggg)ggggg (Qg I N :47SDOccTTTTCC3EccAcTcTcTccA- - ggggg ()Q5S I N :4cccc3EDOcTTccTT— —
Figure imgf000018_0001
T, C or G; n is 0-6; , -TCgTAACgTTgTTTTTAACgTT-3 ' (SEQ ID NO: 9) Ggggggggg)Q ( :72TccTTc3S ID NOccET- - ggggggggg) (Q1cEO :7cTcTTS I NTcTDT - ggggggg)ggg (Q0 NO :7cCATSE IDcTcccTClT— - )gggggggggg (QD NO :69S IcTEccAccAcTcTcTcT3T- - ggggg) (QO :67TTCTCT3SE ID NcAACAAATI - g )gggg (Q058ccccSED N:cTcTcccc3 ITcT— - ggggg) (QO57ccccc3SE IN :TcTDcTcT- -- gggggs (Q"cccc3SE ID §.ccTccTTT_ - ggggsg (Qocc3SE I z"cTcccccDcTTcT- - ggg)gg (QO :5cc ID N4cccccccc3SEcTTccTT- - gggg)ggggg (Qg IN :47SDOccTTTTCC3EccAcTcTcTccA- - ggggg ()Q5S IN :4cccc3EDOcTTccTT —
ggggg)ggg (QO :43c3SE ID NccACAccTAcAT— - gggggg)gg (Q0:42c3SE ID NAATTCTCTcAcTCAACA- I Ggggg)ggg (QO :43c3SE ID NccACAccTAcAT— - gggggg)gg (Q0:42c3SE ID NAATTCTCTcAcTCAACA- I
gggg)gg (Q IO413SED N :AATTTTccAACACCT— - ggggggggggg) (Q :S ID NO40CCATECTCCClT- - ggggg)gg (Q :3cSE ID NO9ccccccccTcTc— - ggggggg)g (Q IO :6SED N3cTCTcTccAAcT - ggggggggggg (QSDE ICcccAccT3T- I Gggg)gg (Q IO413SED N :AATTTTccAACACCT— - ggggggggggg) (Q :S ID NO40CCATECTCCClT- - ggggg)gg (Q :3cSE ID NO9ccccccccTcTc— - ggggggg)g (Q IO :6SED N3cTCTcTccAAcT - ggggggggggg (QSDE ICcccAccT3T- I
gg)gg (QD N :34SE IOCcATTCTT3cTcTAATT— - gggggggggg)gQ (SD N : 33E IOcTTTTTccc — Gg)gg (QD N :34SE IOCcATTCTT3cTcTAATT— - gggggggggg)gQ (SD N : 33E IOcTTTTTccc —
ggggg)ggggQ ( N :SE IDO32cccAC3TcTcAT— —、 Ggggg)ggggQ ( N :SE IDO32cccAC3TcTcAT— —,
ggggggggg) (Q ID NO :31TTSECTTTccTITc- I Ggggggggg) (Q ID NO :31TTSECTTTccTITc- I
gg)ggg (QS ID N0:30Ac3EcccTcTccTTCTc- - gg)gggggg (QO20T3SE ID N :AcTcTcTCAcTTcTcTTTAT- —、 Gg)ggg (QS ID N0:30Ac3EcccTcTccTTCTc- - gg)gggggg (QO20T3SE ID N :AcTcTcTCAcTTcTcTTTAT- —,
gggg ()gggQS7E ID N0:1cccT3ACATAcTTcTcTTC- - gggg)g (Q N IDO: 15TCT3SEcTcTcTTcTT— - gg)gg (Q N IDO :14cSECTTTTTTTCTTcTI - gggQ)gg (gS ID N :2T3EO1cTTTcTTcTTTcT— — ggg)g (Qg IED NO10TT3S :CAcATCTCTATAcT- I ， -TCgTCgACgTCgTTgggCggg-3 ' (SEQ ID NO :73) Gggg ()gggQS7E ID N0:1cccT3ACATAcTTcTcTTC- - gggg)g (QN IDO: 15TCT3SEcTcTcTTcTT_ - gg)gg (QN IDO :14cSECTTTTTTTCTTcTI - gggQ)gg (gS ID N :2T3EO1cTTTcTTcTTTcT— — ggg)g (Qg IED NO10TT3S :CAcATCTCTATAcT- I , -TCgTCgACgTCgTTgggCggg-3 ' (SEQ ID NO:73)
， -TCgCAgTTgTCgTAACgTTgggCggg-3 ' (SEQ ID NO :74) ，一 TCgTCgTTggTATgTT— 3， (SEQ ID NO :77) , -TCgCAgTTgTCgTAACgTTgggCggg-3 ' (SEQ ID NO: 74), a TCgTCgTTggTATgTT-3, (SEQ ID NO: 77)
， - TCgTCgTCgTCgTTgTCgTT- 3， (SEQ ID NO :78) , - TCgTCgTCgTCgTGgTGgTT- 3, (SEQ ID NO: 78)
， -TCgTCgTCgTCgTTgTCgTTgggg-3 ' (SEQ ID NO :79) ， -TCgTTCggggTgCCg-3' (SEQ ID NO :80) , -TCgTCgTCgTCgTTgTCgTTgggg-3 ' (SEQ ID NO:79) , -TCgTTCggggTgCCg-3' (SEQ ID NO:80)
， - TCgTTCggggTAACgATT- 3， (SEQ ID NO :81) , - TCgTTCggggTAACgATT- 3, (SEQ ID NO : 81)
， - TCgTTCggggTAACgTT- 3， (SEQ ID NO :82) , - TCgTTCggggTAACgTT- 3, (SEQ ID NO : 82)
， -TCgTTCggggTACCgAT-3' (SEQ ID NO :83) , -TCgTTCggggTACCgAT-3' (SEQ ID NO:83)
， - TCgTACggCCgCCgTACggCggg- 3， (SEQ ID NO :92) , - TCgTACggCCgCCgTACggCggg- 3, (SEQ ID NO : 92)
， -TCgCgTCgACTCCCCTCgAgggg-3 ' (SEQ ID NO :94) , -TCgCgTCgCCCCCCTCgAgggg-3 ' (SEQ ID NO:94)
， -TCgTCgTCgACTCgTggTCggggg-3 ' (SEQ ID NO :95) ， - TCgggCgCCCgATCgggggg- 3， (SEQ ID NO :96) , -TCgTCgTCgACTCgTggTCggggg-3 ' (SEQ ID NO:95) , - TCgggCgCCCgATCgggggg-3, (SEQ ID NO:96)
， -TCgTCggTCTTTCgAAATT-3 ' (SEQ ID NO :97) , -TCgTCggTCTTTCgAAATT-3 ' (SEQ ID NO :97)
， -TCgTgACgTCCTCgAgTT-3 ' (SEQ ID NO :98) , -TCgTgACgTCCTCgAgTT-3 ' (SEQ ID NO : 98)
， - TCgTCTTTCgACTCgTTCTC- 3， (SEQ ID NO: 115) , - TCgTCTTTCgACTCgTTCTC- 3, (SEQ ID NO: 115)
， -TCgTCgTTTTgCgTTCTC-3 ' (SEQ ID NO: 116) , -TCgTCgTTTTgCgTTCTC-3 ' (SEQ ID NO: 116)
， -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 119) , -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 119)
， -TCgTCgTTTCgTCgTTCTC-3 ' (SEQ ID NO: 120) , -TCgTCgTTTCgTCgTTCTC-3 ' (SEQ ID NO: 120)
， - TCgTCgTCgTCgTTgTCgTT- 3， (SEQ ID NO: 121) , - TCgTCgTCgTCgTGgTGgTT- 3, (SEQ ID NO: 121)
， -TCgTTCTCgACTCgTTCTC-3 ' (SEQ ID NO: 122) , -TCgTTCTCgACTCgTTCTC-3 ' (SEQ ID NO: 122)
， -TCgACgTTCgTCgTTCgTCgTTC-3 ' (SEQ ID NO :123) ， - TCgTCgACgTCgTTCgTTCTC- 3， (SEQ ID NO :124) , -TCgACgTTCgTCgTTCgTCgTTC-3 ' (SEQ ID NO: 123) , - TCgTCgACgTCgTTCgTTCTC-3, (SEQ ID NO: 124)
， - TCgTgCgACgTCgCAgATgAT - 3， (SEQ ID NO: 138), - TCgTgCgACgTCgCAgATgAT - 3, (SEQ ID NO: 138)
' -TCgTCgAgCgCTCgATCggAT-3 ' (SEQ ID NO :141) ' -TCgTCgAgCgCTCgATCggAT-3 ' (SEQ ID NO :141)
， -TCgTCgTTTCgTAgTCgTTgACgTCggg-3' (SEQ ID NO: 142) ， -TCgTCggACgTTTTCCgACgTTCT-3 ' (SEQ ID NO: 144) ， -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 151) ， -TCgTCgTTTgTCgTgTgTCgTT-3； (SEQ ID NO: 152) -TCgTCgTTTCgTAgTCgTGggggggg-3' (SEQ ID NO: 142), -TCgTCggACgTTTTCCgACgTTCT-3 ' (SEQ ID NO: 144), -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 151), -TCgTCgTTTgTCgTgTgTCgTT-3; (SEQ ID NO: 152)
， -TCgTCgTTggTCggggTCgTTggggTCgTT-3' (SEQ ID NO :153) 5， -TCgTCgTTTCgTCTCTCgTT-3 ' (SEQ ID NO : 154) , -TCgTCgTTggTCggggTCgTTggggTCgTT-3' (SEQ ID NO:153) 5, -TCgTCgTTTCgTCTCTCgTT-3 ' (SEQ ID NO: 154)
5， - TCgTCgTTTTgCTgCgTCgTT- 3， (SEQ ID NO : 155)  5, - TCgTCgTTTTgCTgCgTCgTT- 3, (SEQ ID NO: 155)
5， -TCgAgCgTTTTCgCTCgAATT-3 ' (SEQ ID NO : 178)  5, -TCgAgCgTTTTCgCTCgAATT-3 ' (SEQ ID NO: 178)
(5 ) 包含 TTCGTCG的序列 (5) Sequence containing TTCGTCG
5' -TTCgTCgTTTgATCgATgTTCgTTgggggg-3' (SEQ ID NO : 25)  5' -TTCgTCgTTTgATCgATgTTCgTTgggggg-3' (SEQ ID NO: 25)
5， -TTCgTCgTTgTgATCgATgggggg-3 ' -3， (SEQ ID NO : 26)  5, -TTCgTCgTTgTgATCgATgggggg-3 ' -3, (SEQ ID NO : 26)
5， -TATCgATgTTTTCgTCgTCgTTgggggg-3 ' (SEQ ID NO : 27)  5, -TATCgATgTTTTCgTCgTCgTTgggggg-3 ' (SEQ ID NO : 27)
5， -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO : 119)  5, -TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 119)
5， -TCgTCgTTTCgTCgTTCTC-3， (SEQ ID NO : 120)  5, -TCgTCgTTTCgTCgTTCTC-3, (SEQ ID NO: 120)
5， -TCgACgTTCgTCgTTCgTCgTTC-3 ' (SEQ ID NO : 123)  5, -TCgACgTTCgTCgTTCgTCgTTC-3 ' (SEQ ID NO : 123)
5， -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO : 151) 实施例 2 单链脱氧寡核苷酸的合成  5, -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 151) Example 2 Synthesis of single-stranded deoxyoligonucleotides
DNA合成采用固相亚磷酰胺三酯法合成 DNA片段， 此方法具有髙效、 快速偶联等 优点， 已在 DNA化学合成中广泛使用。 DNA synthesis uses a solid phase phosphoramidite triester method to synthesize DNA fragments. This method has the advantages of efficacious and rapid coupling, and has been widely used in DNA chemical synthesis.
DNA化学合成不同于酶促的 DNA合成过程从 5'—3'方向延伸， 而是由 3'端开始。 具体的反应步骤如下：  DNA chemical synthesis differs from the enzymatic DNA synthesis process in that it extends from the 5'-3' direction, but starts at the 3' end. The specific reaction steps are as follows:
一、脱保护基  First, deprotection
用三氯乙酸脱去连结在 CpG (Controlled Pore Glass)上的核苷酸的保护基团 DMT (二甲氧基三苯甲基）， 获得游离的 5' -羟基端， 以供下一步缩合反应。  The protective group DMT (dimethoxytrityl) of the nucleotide attached to CpG (Controlled Pore Glass) was removed with trichloroacetic acid to obtain a free 5'-hydroxyl end for the next condensation reaction. .
二、 活化  Second, activation
将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合并进入合成柱， 形成亚磷酰胺 四唑活性中间体 (其 3' -端已被活化， 但 5' -端仍受 DMT保护）， 此中间体将与 GpG上 的已脱保护基的核苷酸发生缩合反应。  The phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protection), this intermediate will undergo a condensation reaction with a deprotected nucleotide on GpG.
三、 连接  Third, the connection
亚磷酰胺四唑活性中间体遇到 CpG上已脱保护基的核苷酸时， 将与其 5' -羟基发 生亲合反应， 缩合并脱去四唑， 此时合成的寡核苷酸链向前延长一个碱基。  When the phosphoramidite tetrazole active intermediate encounters a deprotected nucleotide on CpG, it will affinity with its 5'-hydroxyl group, condense and remove the tetrazole, and the synthesized oligonucleotide chain Extend one base before.
四、 封闭  Fourth, closed
缩合反应后为了防止连在 CpG上的未参与反应的 5' -经基在随后的循环反应中被 延伸， 常通过乙酰化来封闭此端羟基，一般乙酰化试剂是用乙酸酐和 N-甲基咪唑等混 合形成的。 After the condensation reaction, in order to prevent the 5'-trans group which is not involved in the reaction on CpG from being extended in the subsequent cycle reaction, the terminal hydroxyl group is often blocked by acetylation, and the general acetylation reagent is acetic anhydride and N-methyl. Mixture Formed together.
五、 氧化  V. Oxidation
缩合反应时核苷酸单体是通过亚磷酯键与连在 CpG上的寡核苷酸连接， 而亚磷酯 键不稳定， 易被酸、 碱水解， 此时常用碘的四氢呋喃溶液将亚磷酰转化为磷酸三酯， 得到稳定的寡核苷酸。  In the condensation reaction, the nucleoside monomer is linked to the oligonucleotide attached to CpG through a phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by an acid or a base. At this time, a tetrahydrofuran solution of iodine is usually used. The phosphoryl group is converted to a phosphate triester to obtain a stable oligonucleotide.
经过以上五个步骤后， 一个脱氧核苷酸就被连到 CPG的核苷酸上， 同样再用三氯 乙酸脱去新连上的脱氧核苷酸 5'」经基上的保护基团 DMT后， 重复以上的活化、连接、 封闭、 氧化过程即可得到一 DNA片段粗品。 最后对其进行切割、 脱保护基 (一般对八、 C碱基采用苯甲酰基保护； G碱基用异丁酰基保护； T碱基不必保护； 亚磷酸用腈乙基 保护）、 纯化（常用的有 HAP, PAGE, HPLC, C18, 0PC等方法）、 定量等合成后处理即 可得到符合实验要求的寡核苷酸片段。 After the above five steps, a deoxynucleotide is attached to the nucleotide of C P G, and the deprotected nucleotide of the newly attached deoxynucleotide 5'" is also removed by trichloroacetic acid. After the DMT is repeated, the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment. Finally, it is cleaved and deprotected (generally protected with benzoyl group for octa and C bases; protected with isobutyryl group for G base; T base is not protected; phosphite is protected with nitrile ethyl), purified (commonly used The HAP, PAGE, HPLC, C18, 0PC, etc. methods, quantitative and other synthetic post-treatment can obtain oligonucleotide fragments that meet the experimental requirements.
固相合成寡核苷酸是在 DNA合成仪上进行的， 上述方法合成的寡核苷酸在脱去保 护基后， 目的寡核苷酸纯度是极低的， 含有大量的杂质， 主要杂质有所脱下的保护基 与氨形成的苯甲酸氨和异丁酸氨， 腈磷基上脱下的腈乙基以及合成时产生的短链等， 以至于粗产品中寡核苷酸含量仅为 15%左右。尽管合成时每一步的效率都在 97%〜98%， 但累积的效率并不高。 以链长 20mer和 50mer为例， （97. 5%) 20^60%、 (97. 5%) 50^ 28%, 可见在粗产品中目的寡核苷酸含量很低， 甚至 10%都不到。 这些杂质， 尤其是存 在于粗产品中的大量盐和短链， 造成定量不准， 影响下一步的反应， 因此必须对寡核 苷酸进行纯化。建议采用聚丙烯酰胺凝胶电泳 (PAGE)纯化，该方法纯化的产品纯度高， 可用于绝大部份的分子生物学实验， 可避免许多意想不到的麻烦。 若考虑节约经费， 对于要求较低的实验， 如简单的 PCR反应， 则采用脱盐纯化即可。  The solid phase synthesis oligonucleotide is carried out on a DNA synthesizer. After the deprotection group is removed from the oligonucleotide synthesized by the above method, the purity of the target oligonucleotide is extremely low, and contains a large amount of impurities, and the main impurities are The removed protecting group is ammonia and benzoic acid ammonia and isobutyric acid ammonia, the nitrile ethyl group removed on the nitrile phosphorus group, and the short chain generated during synthesis, so that the amount of the oligonucleotide in the crude product is only About 15%. Although the efficiency of each step in the synthesis is between 97% and 98%, the cumulative efficiency is not high. Taking the chain lengths of 20mer and 50mer as examples, (97.5%) 20^60%, (97.5%) 50^28%, it can be seen that the target oligonucleotide content in the crude product is very low, even 10%. To. These impurities, especially the large amounts of salts and short chains present in the crude product, cause quantitative inaccuracy and affect the next reaction, so the oligonucleotide must be purified. Purification by polyacrylamide gel electrophoresis (PAGE) is recommended. The purified product is highly purified and can be used in most molecular biology experiments to avoid many unexpected problems. If cost savings are considered, for less demanding experiments, such as simple PCR reactions, desalting purification can be used.
寡核苷酸 DNA是以 0D26。值来计量的。在 1ml的 1cm光程标准石英比色皿中， 260nm 波长下吸光度为 1的寡核苷酸溶液定义为 1 0D26。。虽然对于每种特定的寡核苷酸来说， 其碱基的组成不尽相同， 但 1260 0D寡核苷酸 DNA的重量约为 33 g。 实施例 3人工合成的单链脱氧核苷酸对热休克蛋白丙型肝炎病毒抗原肽融合蛋白免疫 刺激作用的增强作用 The oligonucleotide DNA is 0D 26 . The value is measured. Absorbance oligonucleotide solution as defined in 1 0D 26 1, 1cm light path under the standard 1ml quartz cuvette 260nm wavelength. . Although the base composition is not identical for each particular oligonucleotide, the 1260 0D oligonucleotide DNA weighs approximately 33 g. Example 3 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein hepatitis C virus antigen peptide fusion protein
一、 丙型肝炎病毒特异性细胞毒性 Τ淋巴细胞杀伤试验 I. Hepatitis C virus-specific cytotoxicity Τ Lymphocyte killing test
1、 材料： 1. Material:
结核杆菌热休克蛋白 65 (HSP65)和多表位 HCV核心抗原融合蛋白（HSP-HCV) (参 见 CN02122116. 2)， 人工合成的脱氧寡核苷酸（Oligo), 转染 HCV核心抗原多表位抗 原肽基因的 B16细胞 （HCV+B16细胞） （ATCC参照 CN02122116. 2)。 Mycobacterium tuberculosis heat shock protein 65 (HSP65) and multi-epitope HCV core antigen fusion protein (HSP-HCV) See CN02122116. 2), synthetic deoxyoligonucleotide (Oligo), B16 cells (HCV+B16 cells) transfected with the HCV core antigen multi-epitope antigen peptide gene (ATCC reference CN02122116. 2).
所采用的人工合成的单链脱氧核苷酸序列以 Oligo 1-10表示， 其相应序列如序 列表中所示。 （下同）  The synthetic single-stranded deoxynucleotide sequence employed is represented by Oligo 1-10, the corresponding sequences of which are shown in the sequence listing. (the same below)
Oligo 1： SEQ ID N07  Oligo 1: SEQ ID N07
Oligo 2： SEQ ID NO106  Oligo 2: SEQ ID NO106
Oligo 3： SEQ ID NO 103  Oligo 3: SEQ ID NO 103
Oligo 4： SEQ ID NO 18  Oligo 4: SEQ ID NO 18
Oligo 5： SEQ ID N086  Oligo 5: SEQ ID N086
Oligo 6： SEQ ID N079  Oligo 6: SEQ ID N079
Oligo 7： SEQ ID N095  Oligo 7: SEQ ID N095
Oligo 8： SEQ ID NO 123  Oligo 8: SEQ ID NO 123
Oligo 9： SEQ ID N0124  Oligo 9: SEQ ID N0124
Oligo 10： SEQ ID NO 159  Oligo 10: SEQ ID NO 159
2、 实验动物及分组- 采用 6- 8周的雄性 C57/BL6小鼠 （购自北京维通利华实验动物有限公司） 40只， 每组 10只小鼠， 分为生理盐水组 （注射生理盐水）， HSP- HCV组 （注射 20μ§ HSP- HCV)， Oligo组 （注射 50μ§ Oligo), HSP-HCV+01igo组 （注射 20μ§ HSP- HCV, 50μ§ 01igo)。 HSP- HCV和 Oligo分别用 PBS配制成所需浓度， HSP- HCV+Oligo组是将 HSP- HCV和 Oligo 混合后注射。 2, experimental animals and grouping - 6-8 weeks of male C57/BL6 mice (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) 40, each group of 10 mice, divided into saline group (injection physiology) brine), HSP- HCV group (injection 20μ § HSP- HCV), Oligo group (injection 50μ § Oligo), HSP-HCV + 01igo group (injection 20μ § HSP- HCV, 50μ § 01igo ). HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo.
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
于第 1， 14和 21天分别给各组小鼠注射生理盐水、 HSP- HCV应用液、 Oligo应用 液和 HSP- HCV+Oligo混合液。 于小鼠四肢靠近淋巴结处四点皮下注射 （SC)  On the 1st, 14th and 21st day, mice in each group were injected with physiological saline, HSP-HCV application solution, Oligo application solution and HSP-HCV+Oligo mixture. Four subcutaneous injections (SC) in the limbs of the mice near the lymph nodes
(2) 制备效应细胞  (2) Preparation of effector cells
在第 26天将小鼠脱颈处死。 按常规方法取小鼠脾细胞或淋巴结细胞， 制备单细 胞悬液。用含 10% FBS的 IMM培养基重悬细胞，加入含 10% ConA及 HSP-HCV(20( g/ml) 的 IMDM培养基剌激小鼠脾细胞。 37°C， 5% C02培养 7天。 收获小鼠脾细胞做效应细 胞。 (3) 制备靶细胞 The mice were sacrificed by necking on the 26th day. Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension. The cells were resuspended in IMM medium containing 10% FBS, and the mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-HCV (20 (g/ml). 37 ° C, 5% C0 2 culture 7 Days. Mouse spleen cells were harvested for effector cells. (3) Preparation of target cells
采用 10% FBS的 IMDM培养基培养 HCV+B16细胞。用 "Cr标记 HCV+B16细胞（在 37°C， 5% C02条件下培养 1小时）。 用含 10% FBS的 IMDM洗涤细胞， 共三次。 HCV+B16 cells were cultured in 10% FBS in IMDM medium. The cells were incubated with Cr-labeled HCV+B16 cells (1 hour at 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
(4) 细胞毒实验  (4) Cytotoxicity experiment
将 51Cr标记 HCV+B16细胞于 10% FBS的 IMDM培养基中加入到 96孔培养板的孔中， 每孔 ΙΟΟμΙ , 含 1 X 104个细胞。 按 100: 1效靶比加入效应细胞 （ΙΟΟμΙ ), 设三个重复 孔。 37°C， 5% C02培养 4-6小时。 将 96孔培养板离心 5分钟 （3000rpm)。 自每孔吸 出 ΙΟΟμΙ上清， 测放射性。 按下述公式计算效应细胞的细胞毒活性， 以特异性杀伤率 表示。 51 Cr-labeled HCV+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, each well ΙμΙ containing 1×10 4 cells. Three effector cells (ΙΟΟμΙ) were added at a ratio of 100:1 to the target, and three replicate wells were set. Incubate for 4-6 hours at 37 ° C, 5% C0 2 . The 96-well culture plate was centrifuged for 5 minutes (3000 rpm). The ΙΟΟμΙ supernatant was aspirated from each well and the radioactivity was measured. The cytotoxic activity of effector cells was calculated according to the following formula, expressed as specific killing rate.
特异杀伤率 (%) =实验孔 rpm—自发释放 rpm/最大释放 rpm—自发释放 rpm 4、 实验结果：  Specific kill rate (%) = experimental well rpm - spontaneous release rpm / maximum release rpm - spontaneous release rpm 4, experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （n=10)。  The results are shown in the table below, where the data represents the mean of the 10 mouse data (n=10).
表 1-1 人工合成的单链寡核苷酸对 HSP65-HCV诱生特异性 CTL活性的增强作用  Table 1-1 Enhancement of HSP65-HCV-induced specific CTL activity by synthetic single-stranded oligonucleotides
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000024_0001
Figure imgf000025_0001
5、 结论： 5 Conclusion:
人工合成的单链脱氧核苷酸可明显增强热休克蛋白丙型肝炎病毒抗原肽融合蛋白 免疫诱生 HCV核心抗原特异性的细胞毒性 T淋巴细胞的活性 （p<0. 05)。 这种 CTL在 体内可杀伤 HCV感染的细胞。 因此， 人工合成的单链脱氧核苷酸可明显增强热休克蛋 白丙型肝炎病毒抗原肽融合蛋白的抗病毒 （包括但不限于丙型肝炎病毒） 活性。 二、 体内杀伤 HCV感染细胞的实验  Synthetic single-stranded deoxynucleotides significantly enhanced the activity of the heat shock protein hepatitis C virus antigen peptide fusion protein by immunologically inducing HCV core antigen-specific cytotoxic T lymphocytes (p<0.05). This CTL kills HCV-infected cells in vivo. Thus, synthetic single-stranded deoxynucleotides significantly enhance the antiviral (including but not limited to hepatitis C virus) activity of the heat shock protein hepatitis C virus antigen peptide fusion protein. Second, the experiment of killing HCV infected cells in vivo
转染 HCV核心抗原多表位抗原肽基因的 B16细胞 （HCV+B16细胞） 可作为丙型肝 炎病毒感染的细胞模型 （CN02122116. 2)。 观察小鼠抑制这种 B16细胞生长的能力， 可检测注射人工合成单链脱氧核苷酸 （Oligo) 和结核杆菌热休克蛋白 65多表位 HCV 核心抗原融合蛋白刺激小鼠 CTL在体内杀伤感染 HCV细胞的活性。  B16 cells (HCV+B16 cells) transfected with the HCV core antigen multi-epitope antigen peptide gene can be used as a cell model for hepatitis C virus infection (CN02122116. 2). To observe the ability of mice to inhibit the growth of this B16 cell, the injection of synthetic single-stranded deoxynucleotides (Oligo) and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein can stimulate mouse CTL to kill HCV in vivo. Cell activity.
1、 材料：  1. Material:
上述结核杆菌热休克蛋白 65(HSP65)和多表位 HCV核心抗原融合蛋白（HSP- HCV)、 人工合成的单链脱氧核苷酸（Oligo) (同实施例 3)、转染 HCV核心抗原多表位抗原肽 基因的 B16细胞 （HC B16细胞）。  The Mycobacterium tuberculosis heat shock protein 65 (HSP65) and the multi-epitope HCV core antigen fusion protein (HSP-HCV), the synthetic single-stranded deoxynucleotide (Oligo) (same as in Example 3), and the transfection of HCV core antigen B16 cells (HC B16 cells) of the epitope antigen peptide gene.
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6-8周的雄性 C57/BL6小鼠 40只， 每组 10只， 分为生理盐水组 （注射生理 盐水）， HSP- HCV组（注射 20μ§ HSP-HCV), Oligo组（注射 50μ§ Oligo)和 HSP- HCV+Oligo 组 （注射 20μ§ HSP-HCV, 50μ§ Oligo),。 Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-HCV group (20 μ § HSP-HCV), and Oligo group (injected 50 μ § Oligo) and HSP-HCV+Oligo group (20 μ § HSP-HCV, 50 μ § Oligo).
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
HSP-HCV和 Oligo分别用 PBS配制成所需浓度， HSP- HCV+Oligo组是将 HSP- HCV 和 Oligo混合后注射。于第 1， 14和 21天分别给各组小鼠注射生理盐水、 HSP-HCVs Oligo 和 HSP- HCV+01igo。 于第 24天接种 HCV+B16细胞 （1 105个/只）， 注射部位是小鼠右侧 背部皮下。 HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo. Each group of mice was injected with normal saline, HSP-HCVs Oligo on days 1, 14, and 21 And HSP- HCV+01igo. At day 24 HCV + B16 cells were seeded (1105 cells / animal), the injection site was the right dorsal skin.
(2) 取肿瘤并称重  (2) Take the tumor and weigh it
在接种肿瘤后的第 15天， 杀死小鼠取肿瘤并称取肿瘤的重量。  On the 15th day after inoculation of the tumor, the mice were sacrificed to take the tumor and weigh the tumor.
4、 实验结果：  4. Experimental results:
表 1-2 人工合成的单链寡核苷酸对 HSP65-HCV剌激小鼠 CTL在体内杀伤感染 HCV细胞活性的增 强作用  Table 1-2 Synthetic single-stranded oligonucleotide pair HSP65-HCV stimulator mice CTL enhances the activity of HCV cells in vivo
Figure imgf000026_0001
5、 结论
Figure imgf000026_0001
5 Conclusion
人工合成的单链脱氧核苷酸（Oligo)可显著增强结核杆菌热休克蛋白 65多表位 HCV核心抗原融合蛋白剌激小鼠 CTL在体内杀伤感染 HCV细胞的活性 （p〈0. 05)， 其 表现是： 联合应用人工合成的单链寡核苷酸和结核杆菌热休克蛋白 65多表位 HCV核 心抗原融合蛋白的小鼠抑制表达 HCV核心抗原的肿瘤细胞的生长能力显著增强。 实施例 4人工合成的单链脱氧核苷酸对热休克蛋白沙眼衣原体主要外膜蛋白表位抗原 肽融合蛋白诱生特异性 CTL活性的增强作用  Synthetic single-stranded deoxynucleotides (Oligo) can significantly enhance the activity of M. tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein to stimulate mouse CTL to kill HCV cells in vivo (p<0.05), The performance is as follows: The combined use of synthetic single-stranded oligonucleotides and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein inhibits the growth ability of tumor cells expressing HCV core antigen significantly. Example 4 Synthetic single-stranded deoxynucleotides enhance the specificity of CTL activity induced by the heat shock protein Chlamydia trachomatis major outer membrane protein epitope antigen peptide fusion protein
1、 材料：  1. Material:
结核杆菌热休克蛋白 65和沙眼衣原体主要外膜蛋白抗原肽融合蛋白 (HSP65- Chla) (CN02141977. 9, 中国） , 人工合成的单链脱氧核苷酸 （Oligo) (同 实施例 3 ) , 转染沙眼衣原体主要外膜蛋白抗原肽基因的 B16细胞 （Chla 16细胞） (ATCC, 参照 CN02141977. 9)。  Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein (HSP65-Chla) (CN02141977. 9, China), synthetic single-stranded deoxynucleotide (Oligo) (same as in Example 3), B16 cells (Chla 16 cells) of the major outer membrane protein antigen peptide gene of Chlamydia trachomatis (ATCC, see CN02141977. 9).
2、 实验动物及分组：  2. Experimental animals and groupings:
釆用 6-8周的雌性 C57/BL6小鼠 40只， 每组 10只， 分为生理盐水组 （注射生理 盐水）， HSP65- Chla组 （注射 20μδ HSP65- Chla), Oligo 组 （注射 50μ Oligo ), HSP65- Chla +01igo组 (注射 2(^g HSP65- Chla, 50μ§ 01igo)。 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group, divided into normal saline group (injected saline), HSP65-Chla group (injected 20μ δ HSP65-Chla), Oligo group (injected 50μ) Oligo), HSP65-Chla +01igo group (injection 2 (^g HSP65-Chla, 50μ § 01igo).
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
HSP65- Chla和 Oligo分别用 PBS配制成所需浓度， HSP65- Chla+Oligo组是将 HSP65-Chla和 Oligo混合后注射。于第 1， 14和 21天分别给各组小鼠注射生理盐水、 HSP65- Chla、 Oligo 和 HSP65_Chla+01igo。 于小鼠四肢靠近淋巴结处四点皮下注射 (SC)。  HSP65-Chla and Oligo were prepared in PBS at the required concentrations, and HSP65-Chla+Oligo was injected with HSP65-Chla and Oligo. Groups of mice were injected with saline, HSP65-Chla, Oligo and HSP65_Chla+01igo on days 1, 14, and 21, respectively. Four subcutaneous injections (SC) were performed on the limbs of the mice near the lymph nodes.
(2) 制备效应细胞  (2) Preparation of effector cells
在第 26天将小鼠脱颈处死。 按常规方法取小鼠脾细胞或淋巴结细胞， 以下操作 如实施例 1的相关表述。  The mice were sacrificed by necking on the 26th day. Mouse spleen cells or lymph node cells were taken in the usual manner, and the following procedure was as described in Example 1.
(3 ) 制备靶细胞  (3) Preparation of target cells
采用 10% FBS的 IMDM培养基培养 Chla+B16细胞。 用 51Cr标记的 Chla+B16细胞做 靶细胞， 下述操作如实施例 1的相关表述。 Chla+B16 cells were cultured in 10% FBS in IMDM medium. Target cells were seeded with 51 Cr-labeled Chla + B16 cells, and the following procedure was performed as described in Example 1.
(4) 细胞毒实验 将 5lCr标记的 Chla+B16细胞于 10% FBS的 IMDM培养基中加入到 96孔培养板的 孔中， 下述操作和细胞毒的计算如实施例 1的相关表述。 (4) Cytotoxicity experiment 5 l of Cr-labeled Chla + B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, and the following procedure and cytotoxicity calculations were performed as described in Example 1.
4、 实验结果： 4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （n=10)。  The results are shown in the table below, where the data represents the mean of the 10 mouse data (n=10).
表 2 人工合成的单链寡核苷酸对 HSP-Chla诱生特异性 CTL活性的增强作用  Table 2 Enhancement of HSP-Chla-induced specific CTL activity by synthetic single-stranded oligonucleotides
Figure imgf000028_0001
Figure imgf000028_0001
5、 结论  5 Conclusion
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65和沙眼衣原体主 要外膜蛋白抗原肽融合蛋白诱生衣原体特异性 CTL的活性 （p〈0. 05)。 这种 CTL在体内 可杀伤感染衣原体的细胞。 因此， 人工合成的单链脱氧核苷酸可明显增强结核杆菌热 休克蛋白 65和沙眼衣原体主要外膜蛋白抗原肽融合蛋白治疗和预防衣原体感染及其 相关疾病的活性。 实施例 5人工合成的单链脱氧核苷酸对结核杆菌热休克蛋白 65和人前列腺特异性抗原 (Prostate specific antigen, PSA)表位抗原肽融合蛋白免疫刺激作用的增强作 用 一、 人工合成的单链寡核苷酸对 HSP65- HCV诱生特异性 CTL活性的增强作用 Synthetic single-stranded deoxynucleotides significantly enhance Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis The outer membrane protein antigen peptide fusion protein was induced to induce the activity of Chlamydia-specific CTL (p<0.05). This CTL kills Chlamydia-infected cells in vivo. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein in the treatment and prevention of Chlamydia infection and its related diseases. Example 5 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on Mycobacterium tuberculosis heat shock protein 65 and human prostate specific antigen (PSA) epitope antigen peptide fusion protein 1. Synthetic single Enhancement of inducible specific CTL activity by HSP65-HCV by strand oligonucleotide
1、 材料：  1. Material:
结核杆菌热休克蛋白 65 (HSP65) 和人前列腺特异性抗原 （Prostate specific antigen, PSA) 抗原肽融合蛋白 （HSP- PSA) (CN01134935. 2, 中国） ， 人工合成的 单链脱氧核苷酸 （Oligo) (同实施例 3)， 转染 PSA抗原肽基因的 B16细胞 （PSA+B16 细胞） （ATCC参照 CN01134935. 2)。  Mycobacterium tuberculosis heat shock protein 65 (HSP65) and human prostate specific antigen (PSA) antigen peptide fusion protein (HSP-PSA) (CN01134935. 2, China), synthetic single-stranded deoxynucleotides (Oligo) (Similar to Example 3), B16 cells (PSA + B16 cells) transfected with the PSA antigen peptide gene (ATCC reference CN01134935. 2).
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6-8周的雄性 C57/BL6小鼠 40只， 每组 10只， 分为生理盐水组 （注射生 理盐水）， HSP-PSA组（注射 20μ§ HSP- PSA )， 01 i go组（注射 50μβ 01 i go )， HSP-PSA+Oli go 组 (注射 20μ§ HSP-PSA, 50μ§ 01igo)。 Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-PSA group (20 μ § HSP-PSA), 01 i go group (injection) 50μ β 01 i go ), HSP-PSA+Oli go group (20μ § HSP-PSA, 50μ § 01igo).
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
于第 1 , 14 和 21 天分别给各组小鼠注射生理盐水、 HSP- PSA、 Oligo 和 HSP-PSA+Oli go o 于小鼠四肢靠近淋巴结处四点皮下注射 （SC；)。  On the 1st, 14th and 21st day, mice in each group were injected with normal saline, HSP-PSA, Oligo and HSP-PSA+Oli go o in four subcutaneous injections (SC;) in the limbs of the mice near the lymph nodes.
(2) 制备效应细胞  (2) Preparation of effector cells
在第 26 天将小鼠脱颈处死。 按常规方法取小鼠脾细胞或淋巴结细胞， 制备单细 胞悬液。用含 10% FBS的 IMDM培养基重悬细胞，加入含 10% ConA及 HSP_PSA(20(^g/ml ) 的 IMDM培养基刺激小鼠脾细胞。 37°C， 5% C02培养 7天。 收获细胞做效应细胞。 The mice were sacrificed by necking on the 26th day. Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension. The cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP_PSA (20 (^g/ml). Cultured at 37 ° C, 5% CO 2 for 7 days. Harvest cells for effector cells.
(3)制备靶细胞  (3) Preparation of target cells
采用含 10% FBS的 IMDM培养基培养转染 PSA抗原肽基因的 B16细胞 （PSA+B16细 胞）。用 61Cr标记 PSA+B16细胞， （培养 1小时， 37X, 5% C02)。 用含 10% FBS的 IMDM 洗涤细胞， 共三次。 B16 cells (PSA + B16 cells) transfected with the PSA antigen peptide gene were cultured in IMDM medium containing 10% FBS. PSA+B16 cells were labeled with 61 Cr (cultured for 1 hour, 37X, 5% C0 2 ). Use IMDM with 10% FBS Wash the cells three times in total.
(4) 细胞毒实验  (4) Cytotoxicity experiment
将 51Cr标记 PSA 16细胞于 10% FBS的 IMDM培养基中加入到 96孔培养板的孔中， 每孔 100μ1， 含 1 X 104个细胞。 按 100 : 1效靶比加入效应细胞， 设三复孔。 下述操作 和细胞毒的计算如实施例 1的相关表述。 51 Cr-labeled PSA 16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 μl per well, containing 1×10 4 cells. The effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
4、 实验结果： 4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （η=10)。 表 3-1 人工合成的单链寡核苷酸对 HSP- PSA诱生特异性 CTL活性的增强作用  The results are shown in the table below, where the data represents the mean of the 10 mouse data (η = 10). Table 3-1 Enhancement of HSP-PSA-inducing specific CTL activity by synthetic single-stranded oligonucleotides
Figure imgf000030_0001
Figure imgf000030_0001
5、 结论： 5 Conclusion:
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65与 PSA抗原肽融 合蛋白免疫诱生 PSA特异性 CTL的活性 （p<0. 05), 这种 CTL可杀伤表达 PSA 的肿瘤 细胞。 因此， 人工合成的单链脱氧核苷酸可明显增强热休克蛋白 PSA抗原肽融合蛋白 预防和治疗前列腺癌的生物学活性。 二、 人工合成的单链寡核苷酸对 HSP65- PSA抗表达 PSA肿瘤活性的增强作用 Synthetic single-stranded deoxynucleotides can significantly enhance the activity of PSK-specific CTLs induced by Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (p<0.05). This CTL can kill PSA-expressing tumors. cell. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the heat shock protein PSA antigen peptide fusion protein. Prevention and treatment of the biological activity of prostate cancer. 2. Enhancement of anti-expression of PSA tumor by HSP65-PSA by synthetic single-stranded oligonucleotide
1、 材料：  1. Material:
上述结核杆菌热休克蛋白 65(HSP65)和人前列腺特异性抗原 (Prostate specific antigen, PSA) 抗原肽融合蛋白 （HSP-PSA)、 人工合成的单链脱氧核苷酸 （Oligo) (同实施例 3)、 转染 PSA抗原肽基因的 B16细胞 （PSA+B16细胞）。  The Mycobacterium tuberculosis heat shock protein 65 (HSP65) and human prostate specific antigen (PSA) antigen peptide fusion protein (HSP-PSA), synthetic single-stranded deoxynucleotide (Oligo) (same as in Example 3 ), B16 cells (PSA + B16 cells) transfected with the PSA antigen peptide gene.
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6-8周的雄性 C57/BL6小鼠 40只， 每组 10只， 分为生理盐水组 （注射生 理盐水）， HSP- PSA组（注射 20μ§ HSP- PSA)， Oligo组 (注射 50μ§ Oligo), HSP-PSA+01igo 组 （注射 20 g HSP-PSA, 50μ§ 01igo)。 Forty male C57/BL6 mice, 6-8 weeks old, were divided into normal saline group (injected with normal saline), HSP-PSA group (20 μ § HSP-PSA), and Oligo group (injected 50 μ § Oligo), HSP-PSA+01igo group (20 g HSP-PSA, 50 μ§ 01igo).
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
HSP- PSA和 Oligo用 PBS配制， HSP- PSA+Oligo组是将 HSP- PSA和 Oligo混合后 注射。于第 1， 14和 21天分别给小鼠注射生理盐水、 HSP-PSA、01igo和 HSP_PSA+01igo。 于第 28天接种 PSA+B16细胞 （1 X 105/只）， 注射部位是小鼠右后背部皮下。 HSP-PSA and Oligo were prepared in PBS, and HSP-PSA+Oligo group was injected with HSP-PSA and Oligo. Mice were injected with saline, HSP-PSA, 01igo and HSP_PSA+01igo on days 1, 14, and 21, respectively. PSA+B16 cells (1×10 5 /piece) were inoculated on the 28th day, and the injection site was subcutaneous in the right back of the mouse.
(2) 取肿瘤并称童  (2) taking a tumor and calling it a child
在接种肿瘤后的第 15天， 杀死小鼠取肿瘤并称肿瘤的重量。  On the 15th day after the tumor was inoculated, the mice were sacrificed to take the tumor and weighed the tumor.
4、 实验结果：  4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （n=10)。 表 3-2人工合成的单链寡核苷酸对 HSP65-PSA抗表达 PSA肿瘤活性的增强作用  The results are shown in the table below, where the data represents the mean of the 10 mouse data (n=10). Table 3-2 Synthetic effect of synthetic single-stranded oligonucleotides on HSP65-PSA anti-expression PSA tumor activity
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000031_0001
Figure imgf000032_0001
5、 结论  5 Conclusion
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65 与 PSA抗原肽融 合蛋白抑制表达 PSA肿瘤细胞的生长（p<0.05)。 人工合成的单链脱氧核苷酸可明显增 强热休克蛋白与 PSA抗原肽融合蛋白预防和治疗前列腺癌的生物学活性。 实施例 6人工合成的单链脱氧核苷酸对热休克蛋白 -MUC1抗原肽融合蛋白免疫刺激作 用的增强作用 一、 人工合成的单链寡核苷酸对 HSP65- MUC1诱生特异性 CTL活性的增强作用  Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (P<0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of heat shock proteins and PSA antigen peptide fusion proteins in the prevention and treatment of prostate cancer. Example 6 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein-MUC1 antigen peptide fusion protein 1. Synthesis of specific CTL activity by HSP65-MUC1 induced by synthetic single-stranded oligonucleotide Enhancement
1、 材料- 结核杆菌热休克蛋白 65 (HSP65) 和 MUC1 抗原肽融合蛋白 （HSP-MUC1) (CN01102614.6, 中国） ， 人工合成的单链脱氧核苷酸 (Oligo) (同实施例 3)， 转染 WJC1抗原肽基因的 B16细胞 （MUC1+B16细胞） （ATCC参照 CN01102614.6)。  1. Materials - Mycobacterium tuberculosis heat shock protein 65 (HSP65) and MUC1 antigen peptide fusion protein (HSP-MUC1) (CN01102614.6, China), synthetic single-stranded deoxynucleotides (Oligo) (same as in Example 3) B16 cells (MUC1+B16 cells) transfected with the WJC1 antigen peptide gene (ATCC reference CN01102614.6).
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6-8周的雌性 C57/BL6小鼠 40只， 每组 10只， 分为生理盐水组 （注射生 理盐水）， HSP-MUC1 组 （注射 20μ§ HSP-MUCl), Oligo 组 （注射 50μ6 Oligo), HSP- MUCl+Oligo组 （注射 20μ§ HSP-MUCl, 50μ§ 01igo)。 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group were divided into normal saline group (injected saline), HSP-MUC1 group (20 μ § HSP-MUCl injection), Oligo group (injected 50 μ 6) Oligo), HSP- MUCl+Oligo group (20 μ § HSP-MUCl, 50 μ § 01igo).
3、 实验方法  3. Experimental methods
(1) 注射  (1) Injection
HSP-MUCl和 Oligo用 PBS配制至所需浓度， HSP- MUCl+Oligo组是将 HSP- MUC1 和 Oligo混合后注射。 于第 1， 14和 21天分别给各组小鼠注射生理盐水、 HSP- MUC1、 Oligo和 HSP- MUCl+01igo。 于小鼠四肢靠近淋巴结处四点皮下注射 （SC)。 (2) 制备效应细胞 HSP-MUCl and Oligo were prepared in PBS to the desired concentration, and the HSP-MUCl+Oligo group was injected with HSP-MUC1 and Oligo. Each group of mice was injected with physiological saline, HSP-MUC1, Oligo and HSP-MUCl+01igo on days 1, 14, and 21. Four subcutaneous injections (SC) were performed on the limbs of the mice near the lymph nodes. (2) Preparation of effector cells
在第 26天将小鼠脱颈处死。按常规方法取小鼠脾细胞或淋巴结细胞， 制备单细胞 悬液。用含 10% FBS的 IMDM培养基重悬细胞，加入含 10% ConA及 HSP- MUC1 (200 g/ral ) 的 IMDM培养基刺激小鼠脾细胞。 37°C, 5% C02培养 7天。 收获细胞做效应细胞。 The mice were sacrificed by necking on the 26th day. Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension. The cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-MUC1 (200 g/ral). Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
( 3) 制备靶细胞  (3) Preparation of target cells
采用 10% FBS的 IMDM培养基培养 MUC1+B16细胞。 用 51Cr标记 PSA+B16细胞 （培 养 1小时， 37°C， 5% C02) o 用含 10% FBS的 IMDM洗涤细胞， 共三次。 MUC1+B16 cells were cultured in 10% FBS in IMDM medium. PSA+B16 cells were labeled with 51 Cr (1 hour, 37 ° C, 5% C0 2 ) o The cells were washed three times with IMDM containing 10% FBS.
(4) 细胞毒实验  (4) Cytotoxicity experiment
将 51Cr标记 UC1+B16细胞于 10% FBS的 IMDM培养基中加入到 96孔培养板的孔 中， 每孔 100μ1， 含 1 X 104个细胞。 按 100 : 1效靶比加入效应细胞， 设三复孔。 下述 操作和细胞毒的计算如实施例 1的相关表述。 51 Cr-labeled UC1+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 μl per well, containing 1×10 4 cells. The effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
4、 实验结果：  4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （η=10)。  The results are shown in the table below, where the data represents the mean of the 10 mouse data (η = 10).
表 4-1 人工合成的单链寡核苷酸对 HSP65-MUC1诱生特异性 CTL活性的增强作用  Table 4-1 Enhancement of inducible specific CTL activity by HSP65-MUC1 by synthetic single-stranded oligonucleotides
Figure imgf000033_0001
5、 结论:
Figure imgf000033_0001
5 Conclusion:
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65- MUC1抗原肽融 合蛋白诱生 MUC1特异性细胞毒性 T淋巴细胞的活性 （p〈0. 05), 这种 CTL可杀伤表达 UC1的肿瘤细胞。 因此， 人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65- MUC1抗原肽融合蛋白预防和治疗表达 MUC1肿瘤如乳腺癌、 卵巢癌、 肺癌、 前列 腺癌、 结直肠癌等的生物学活性。 二、 人工合成的单链寡核苷酸对 HSP65-MUC1抗表达 MUC1肿瘤活性的增强作用 1、 材料- 上述结核杆菌热休克蛋白 65 (HSP65) 和 MUC1抗原肽融合蛋白 （HSP-MUC1 )、 人 工合成的单链脱氧核苷酸 （Oligo) (同实施例 3)、 转染 MUC1抗原肽基因的 B16细胞 (MUC1+B16细胞）。  Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein to induce MUC1-specific cytotoxic T lymphocytes (p<0.05), this CTL can kill expression Tumor cells of UC1. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the prevention and treatment of MUC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. by the Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein. Learning activity. Second, the synthetic single-stranded oligonucleotide enhances the anti-expression of MUC1 tumor activity by HSP65-MUC1. 1. Materials - Mycobacterium tuberculosis heat shock protein 65 (HSP65) and MUC1 antigen peptide fusion protein (HSP-MUC1), artificial Synthetic single-stranded deoxynucleotides (Oligo) (same as in Example 3), B16 cells (MUCl + B16 cells) transfected with the MUC1 antigen peptide gene.
2、 实验动物及分组： 采用 6-8周的雌性 C57/BL6小鼠， 分为生理盐水组 （注射 生理盐水）， HSP-MUC1 组 （注射 20μ§ HSP-MUC1 ), Oligo 组 （注射 50μ§ Oligo), HSP-MUCl+Oligo组 （注射 20μ§ HSP-MUC1 , 50μ§ Oligo), 每组 10只。 2. Experimental animals and grouping: Female C57/BL6 mice were used for 6-8 weeks, divided into normal saline group (injected saline), HSP-MUC1 group (20 μ § HSP-MUC1 injection), Oligo group (injected 50 μ § Oligo), HSP-MUCl+Oligo group (20 μ § HSP-MUC1, 50 μ § Oligo), 10 in each group.
3、 实验方法  3. Experimental methods
( 1 )注射  (1) injection
于第 28天接种 MUC1+B16细胞 ( I x lO5/只）， 注射部位是小鼠右后背部皮下。MUC1+B16 cells (I x lO 5 /only) were inoculated on the 28th day, and the injection site was subcutaneously in the right hind back of the mouse.
(2) 取肿瘤并称重 (2) Take the tumor and weigh it
在接种肿瘤后的第 15天， 杀死小鼠并称肿瘤的重量。  On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
4、 实验结果：  4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （n=10)。 表 4- 2 人工合成的单链寡核苷酸对 HSP65-MUC1抗表达 MUC1肿瘤活性的增强作用 The results are shown in the table below, where the data represents the mean of the 10 mouse data ( n = 10). Table 4-2 Enhancement of anti-expression of MUC1 tumor by HSP65-MUC1 by synthetic single-stranded oligonucleotide
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000034_0001
Figure imgf000035_0001
5、 结论  5 Conclusion
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65- MUC1抗原肽融 合蛋白抑制表达 MUC1肿瘤细胞的生长 （p<0. 05 )。 人工合成的单链脱氧核苷酸可明显 增强结核杆菌热休克蛋白 65-MUC1抗原肽融合蛋白预防和治疗表达 UC1肿瘤如乳腺 癌、 卵巢癌、 肺癌、 前列腺癌、 结直肠癌等的生物学活性。 实施例 7 人工合成的单链脱氧核苷酸对热休克蛋白 HER2抗原肽融合蛋白免疫刺激作 用的增强作用  Synthetic single-stranded deoxynucleotides significantly enhanced the growth of MUC1 tumor cells inhibited by Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein (p<0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein for the prevention and treatment of UC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. . Example 7 Enhancement of immunostimulatory effects of synthetic single-stranded deoxynucleotides on heat shock protein HER2 antigen peptide fusion protein
―、 人工合成的单链寡核苷酸对 HSP65- HER- 2诱生特异性 CTL活性的增强作用―, Enhancement of inducible specific CTL activity by HSP65- HER-2 induced by synthetic single-stranded oligonucleotides
1、 材料- 结核杆菌热休克蛋白 65和 HER-2抗原肽融合蛋白（HSP-HER-2) (CN01136347. 9, 中国） ， 人工合成的单链脱氧核苷酸（Oligo) (同实施例 3)， 转染 HER-2抗原肽基因 的 B16细胞 （HER- 2+B16细胞） (ATCC, 参照 CN01136347. 9)。 1. Materials - Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion protein (HSP-HER-2) (CN01136347. 9, China), synthetic single-stranded deoxynucleotide (Oligo) (same as in Example 3 ), B16 cells (HER- 2+B16 cells) transfected with the HER-2 antigen peptide gene (ATCC, see CN01136347. 9).
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6-8周的雌性 C57/BL6小鼠， 分为生理盐水组（注射生理盐水）， HSP- HER - 2 组 （注射 20μ§ HSP- HER - 2), Oligo组 （注射 50μ Oligo), HSP- HER- 2+01 i go组 （注 射 20 g HSP-HER-2, 50 g Oligo), 每组 10只。 6-8 weeks of female C57/BL6 mice were divided into normal saline group (injected saline), HSP-HER-2 group (20 μ § HSP-HER - 2 injection), Oligo group (50 μ Oligo injection), HSP - HER- 2+01 i go group (injected 20 g HSP-HER-2, 50 g Oligo), 10 in each group.
3、 实验方法  3. Experimental methods
( 1 ) 注射 HSP- HER- 2和 Oligo用 PBS配制， HSP-HER- 2+01igo是将 HSP- HER- 2和 Oligo混 合后注射。 于第 1， 14 和 21 天分别给小鼠注射生理盐水、 HSP-HER- 2、 Oligo 和 HSP-HER-2+OligOo 于小鼠四肢靠近淋巴结处四点皮下注射 （SC)。 (1) injection HSP-HER-2 and Oligo were formulated in PBS, and HSP-HER-2+01igo was prepared by mixing HSP-HER-2 with Oligo. On days 1, 14, and 21, mice were injected with saline, HSP-HER-2, Oligo, and HSP-HER-2+OligOo at four subcutaneous injections (SC) in the limbs of the mice near the lymph nodes.
(2) 制备效应细胞  (2) Preparation of effector cells
在第 26天将小鼠脱颈处死。 按常规方法取小鼠脾细胞或淋巴结细胞， 制备单细 胞悬液。 用含 10% FBS 的 IMDM培养基重悬细胞， 加入含 10% ConA及 HSP- HER-2 (200μβ ) 的 IMDM培养基刺激小鼠脾细胞。 37°C, 5% C02培养 7天。 收获细胞做 效应细胞。 The mice were sacrificed by necking on the 26th day. Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension. With IMDM medium containing 10% FBS, cells were resuspended, IMDM medium containing 10% ConA and HSP- HER-2 (200μ β) stimulation of mouse spleen cells was added. Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
(3) 制备靶细胞  (3) Preparation of target cells
采用 10% FBS的 IMDM培养基培养转染 HER- 2抗原肽基因的 B16细胞 （HER- 2 16 细胞）。 用 51Cr标记 HER-2+B16细胞 （培养 1小时， 37°C, 5% C02)。 用含 10% FBS的 IMDM洗涤细胞， 共三次。 B16 cells (HER-216 cells) transfected with the HER-2 antigen peptide gene were cultured in 10% FBS in IMDM medium. HER-2+B16 cells were labeled with 51 Cr (cultured for 1 hour, 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
(4) 细胞毒实验  (4) Cytotoxicity experiment
将 51Cr标记冊1^-2 16细胞于 10% FBS的 IMDM培养基中加入到 96孔培养板的孔 中， 每孔 100μ1， 含 1X104个细胞。 按 100:1效靶比加入效应细胞， 设三复孔。 51 Cr-labeled 1^-2 16 cells were added to 10% FBS in IMDM medium to wells of a 96-well culture plate, 100 μl per well, containing 1 ×10 4 cells. The effector cells were added at a target ratio of 100:1, and three replicate wells were set.
下述操作和细胞毒的计算如实施例 1的相关表述。  The following operations and cytotoxicity calculations are as described in relation to Example 1.
4、 实验结果： 4. Experimental results:
结果见下表， 其中的数据代表的 10只老鼠数据的均值 （η=10)。 表 5-1 人工合成的单链寡核苷酸对 HSP65- HHER- 2诱生特异性 CTL活性的增强作用  The results are shown in the table below, where the data represents the mean of the 10 mouse data (η = 10). Table 5-1 Enhancement of HSP65-HHER-2 Inducible Specific CTL Activity by Synthetic Single-Stranded Oligonucleotides
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000036_0001
Figure imgf000037_0001
5、 结论：  5 Conclusion:
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白与 HER- 2抗原肽融 合蛋白免疫诱生 HER- 2特异性细胞毒性 T淋巴细胞的活性 （p<0. 05)， 这种 CTL可杀 伤表达 HER-2的肿瘤细胞。 因此， 人工合成的单链脱氧核苷酸可明显增强结核杆菌热 休克蛋白 65与 HER - 2抗原肽融合蛋白预防和治疗表达 HER - 2肿瘤如乳腺癌和卵巢癌 等的生物学活性。 二、 人工合成的单链寡核苷酸对 HSP65- HER-2抗表达 HER-2肿瘤活性的增强作用 Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein and HER-2 antigen peptide fusion protein to induce HER-2 specific cytotoxic T lymphocytes (p<0.05). CTL can kill tumor cells expressing HER-2. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer. Second, the synthetic single-stranded oligonucleotide enhances the anti-expression of HSP65-HER-2 by HER-2 tumor activity
1、 材料： 1. Material:
上述结核杆菌热休克蛋白 65 (HSP65) 和 HER- 2抗原肽融合蛋白 (HSP- HER- 2)、 人工合成的单链脱氧核苷酸（Oligo) (同实施例 3)、转染 HER- 2抗原肽基因的 B16细 胞 （HER-2+B16细胞）。  The above Mycobacterium tuberculosis heat shock protein 65 (HSP65) and HER-2 antigen peptide fusion protein (HSP-HER-2), synthetic single-stranded deoxynucleotide (Oligo) (same as in Example 3), transfected HER-2 B16 cells (HER-2+B16 cells) of the antigenic peptide gene.
2、 实验动物及分组：  2. Experimental animals and groupings:
采用 6- 8周的雌性 C57/BL6小鼠， 分为生理盐水组（注射生理盐水）， HSP- HER-2 组 （注射 20μ8 HSP- HER- 2)， Oligo组 (注射 50μ§ Oligo), HSP- HER- 2+01 i go组 （注 射 20μ§ HSP- HER- 2， 50μ Oligo), 每组 10只。 Six to eight weeks of female C57/BL6 mice were divided into normal saline group (injected with normal saline), HSP-HER-2 group (injected with 20 μ 8 HSP-HER-2), and Oligo group (injected with 50 μ § Oligo). HSP-HER- 2+01 i go group (20 μ § HSP-HER- 2, 50 μ Oligo), 10 in each group.
3、 实验方法  3. Experimental methods
( 1 ) 注射  (1) injection
于第 1， 14 和 21 天分别给小鼠注射生理盐水、 HSP- HER- 2、 Oligo 和 HSP- HER-2+01igo。 于第 28天接种 HER- 2+B16细胞 （1 X 105/只）， 注射部位是小鼠右 后背部皮下。 Mice were injected with saline, HSP-HER-2, Oligo and HSP-HER-2+01igo on days 1, 14, and 21, respectively. On day 28, HER-2+B16 cells (1×10 5 /piece) were inoculated, and the injection site was subcutaneous in the right back of the mouse.
(2) 取肿瘤并称重  (2) Take the tumor and weigh it
在接种肿瘤后的第 15天， 杀死小鼠并称肿瘤的重量。  On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
4、 实验结果： 结果见下表， 其中的数据代表的 10只老鼠数据的均值 （n=10)。 表 5-2人工合成的单链寡核苷酸对 HSP65-HER-2抗表达 HER- 2肿瘤活性的增强作用
Figure imgf000038_0001
4. Experimental results: The results are shown in the table below, where the data represents the mean of 10 mouse data (n=10). Table 5-2 Enhancement of HSP65-HER-2 Anti-Expression of HER-2 Tumor Activity by Synthetic Single-Stranded Oligonucleotides
Figure imgf000038_0001
5、 结论：  5 Conclusion:
人工合成的单链脱氧核苷酸可明显增强结核杆菌热休克蛋白 65与 HER- 2抗原肽 融合蛋白抑制表达 HER-2肿瘤细胞的生长 （p<0. 05)。 人工合成的单链脱氧核苷酸可明 显增强结核杆菌热休克蛋 S 65与 HER-2抗原肽融合蛋白预防和治疗表达 HER- 2肿瘤如 乳腺癌和卵巢癌等的生物学活性。  Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins (P<0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock egg S 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer.

Claims

权 利 要 求 、 一种疫苗组合物， 其特征在于包含人工合成的单链脱氧核苷酸、 抗原或抗原组合 物， 所述人工合成的单链脱氧核苷酸增强所述抗原或抗原组合物的免疫剌激作用。 、 权利要求 1 的疫苗组合物， 其中所述的人工合成的单链脱氧核苷酸包含至少一个 选自下式之一的序列- Claims, a vaccine composition, characterized by comprising a synthetic single-stranded deoxynucleotide, antigen or antigen composition, said synthetic single-chain deoxynucleotide enhancing immunity of said antigen or antigen composition Stimulating effect. The vaccine composition of claim 1, wherein said synthetic single-stranded deoxynucleotide comprises at least one sequence selected from the group consisting of -
( 1 ) (G)n(L)n X1X2CGY1Y2(M)n (G)n, 其中 为入， T或 G; X2为 A或 T; Yi为 A 或 T; Y2.为 A， T或 C; L和 M分别为 A， T， C或 G; n为 0-6的整数；(1) (G) n (L) n X 1 X 2 CGY 1 Y 2 (M) n (G) n , where is in, T or G; X 2 is A or T; Yi is A or T; Y 2. A, T or C; L and M are respectively A, T, C or G; n is an integer from 0 to 6;
(2) (G)n(L)„CG(XY)nCG(M)„(G)n, 其中 X为 A或 T; Υ为 Α或 T; L和 Μ分别 为 A， T， C或 G; n为 0-6的整数； (2) (G) n (L) „CG(XY) n CG(M)„(G)n, where X is A or T ; Υ is Α or T; L and Μ are A, T, C or G; n is an integer from 0-6;
(3 ) (TCG)n(L)„CG (M)n(G)n， 其中 L和 M分别为 A， T， C或 G; n为 0-6的整数；(3) (TCG) n (L) „ CG (M) n (G) n , where L and M are respectively A, T, C or G; n is an integer from 0 to 6;
(4) (TCG)„(L)nX1X2CG (M)n, 其中 为 A， T或 G; X2为 A或 T; L和 M分别为 A, T， C或 G; n为 0-6的整数； (4) (TCG) „(L) n X 1 X 2 CG (M)n, where A, T or G; X 2 is A or T; L and M are A, T, C or G, respectively; An integer of 0-6;
( 5 ) 包含 TTCGTCG的序列。  (5) A sequence containing TTCGTCG.
、 权利要求 2所述的疫苗组合物， 其中所述的人工合成的单链脱氧核苷酸选自 SEQ ID NO: 1-180任一所示的序列。 The vaccine composition according to claim 2, wherein the artificially synthesized single-stranded deoxynucleotide is selected from the ones shown in any one of SEQ ID NOS: 1-180.
、 权利要求 2或 3所述的疫苗组合物， 其中所述的人工合成的单链脱氧核苷酸在其 碱基上各个基团被修饰。 The vaccine composition according to claim 2 or 3, wherein the artificially synthesized single-stranded deoxynucleotide is modified at each of its bases.
、 权利要求 4所述的疫苗组合物， 其中所述的修饰包括非硫代修饰、 硫代修饰、 部 分硫代修饰、稀有碱基修饰、 甲基化修饰、巯基、 Aminolinker C6、或 Thiol-C6 S-S 用于与其他物质偶联所应用的修饰方式。 The vaccine composition according to claim 4, wherein the modification comprises a non-thio modification, a thio modification, a partial thio modification, a rare base modification, a methylation modification, a thiol group, an Aminolinker C6, or a Thiol-C6 The modification used by SS for coupling to other substances.
、 权利要求 1 所述的疫苗组合物， 其中所述的抗原或抗原组合物为热休克蛋白与抗 原肽融合所形成的融合蛋白。 The vaccine composition according to claim 1, wherein the antigen or antigen composition is a fusion protein formed by fusion of a heat shock protein and an antigen peptide.
、 按照权利要求 6所述的疫苗组合物， 其中所述的热休克蛋白为结核杆菌热休克蛋 白 65 The vaccine composition according to claim 6, wherein said heat shock protein is Mycobacterium tuberculosis heat shock protein 65
、 按照权利要求 6或 7所述的疫苗组合物， 其中所述的抗原肽选自多表位丙型肝炎 病毒核心抗原肽， 沙眼衣原体主要外膜蛋白表位抗原肽， 人前列腺特异性抗原肽， MUC1抗原肽或 HER-2抗原肽。 The vaccine composition according to claim 6 or 7, wherein the antigenic peptide is selected from the group consisting of a multi-epitope hepatitis C virus core antigen peptide, a Chlamydia trachomatis major outer membrane protein epitope antigen peptide, and a human prostate specific antigen peptide. , MUC1 antigen peptide or HER-2 antigen peptide.
、 一种生产权利要求 1所述的疫苗组合物的方法， 包括将权利要求 2至 5任一项所 述的单链脱氧核苷酸与抗原或抗原组合物混合。 A method of producing the vaccine composition of claim 1, comprising the method of any one of claims 2 to 5. The single-stranded deoxynucleotides are mixed with an antigen or antigen composition.
、 权利要求 9所述的方法， 其中所述抗原或抗原组合物为热休克蛋白与抗原肽 融合所形成的融合蛋白。 The method of claim 9, wherein the antigen or antigen composition is a fusion protein formed by fusion of a heat shock protein and an antigen peptide.
、 权利要求 10所述的方法， 其中所述的热休克蛋白为结核杆菌热休克蛋白 65。 、 权利要求 10或 11所述的方法， 其中所述的抗原肽选自多表位丙型肝炎病毒 核心抗原肽，沙眼衣原体主要外膜蛋白表位抗原肽，人前列腺特异性抗原肽, MUC1 抗原肽或 HER-2抗原肽。 The method of claim 10, wherein the heat shock protein is Mycobacterium tuberculosis heat shock protein 65. The method according to claim 10 or 11, wherein the antigenic peptide is selected from the group consisting of a multi-epitope hepatitis C virus core antigen peptide, a Chlamydia trachomatis major outer membrane protein epitope antigen peptide, a human prostate specific antigen peptide, and a MUC1 antigen. Peptide or HER-2 antigen peptide.
、 一种增强抗原或抗原组合物免疫刺激作用的方法， 包括将权利要求 2至 5任 一项所述的单链脱氧核苷酸与抗原或抗原组合物联合应用。 A method of enhancing immunostimulatory action of an antigen or antigen composition, comprising the use of the single-chain deoxynucleotide of any one of claims 2 to 5 in combination with an antigen or antigen composition.
、 权利要求 13述的方法， 其中所述抗原或抗原组合物为热休克蛋白与抗原肽融 合所形成的融合蛋白。 The method of claim 13, wherein the antigen or antigen composition is a fusion protein formed by fusion of a heat shock protein and an antigen peptide.
、 权利要求 14所述的方法， 其中所述的热休克蛋白为结核杆菌热休克蛋白 65。 、 权利要求 14或 15所述的方法， 其中所述的抗原肽选自多表位丙型肝炎病毒 核心抗原肽，沙眼衣原体主要外膜蛋白表位抗原肽，人前列腺特异性抗原肽， MUC1 抗原肽或 HER-2抗原肽。 The method of claim 14, wherein the heat shock protein is Mycobacterium tuberculosis heat shock protein 65. The method according to claim 14 or 15, wherein the antigenic peptide is selected from the group consisting of a multi-epitope hepatitis C virus core antigen peptide, a Chlamydia trachomatis major outer membrane protein epitope antigen peptide, a human prostate specific antigen peptide, and a MUC1 antigen. Peptide or HER-2 antigen peptide.
、 权利要求 1至 8任一项所述的疫苗组合物在制备用于治疗病毒感染、 细菌感 染、 寄生虫感染、 变态反应或癌症的疫苗中的应用。 Use of the vaccine composition according to any one of claims 1 to 8 for the preparation of a vaccine for the treatment of a viral infection, a bacterial infection, a parasitic infection, an allergy or cancer.
、 权利要求 17所述的应用， 其中所述疫苗组合物的应用对象为人类或哺乳类动 物。 The use according to claim 17, wherein the vaccine composition is applied to a human or a mammalian animal.
、 权利要求 17所述的用途， 其中所述的病毒感染为丙型肝炎病毒感染或由该病 毒感染引起的相关疾病。 The use according to claim 17, wherein the viral infection is a hepatitis C virus infection or a related disease caused by the infection of the virus.
、 权利要求 17所述的用途， 其中所述的细菌感染为衣原体感染或由衣原体感染 引起的相关疾病。 The use according to claim 17, wherein the bacterial infection is a Chlamydia infection or a related disease caused by Chlamydia infection.
、 权利要求 17所述的用途， 其中所述的癌症为前列腺癌、 乳腺癌、 卵巢癌、 肺 癌、 胃癌、 子宫内膜癌、 唾液腺癌、 腺癌、 结肠和直肠癌、 非小细胞肺癌或肺腺 癌。 The use according to claim 17, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, gastric cancer, endometrial cancer, salivary gland cancer, adenocarcinoma, colon and rectal cancer, non-small cell lung cancer or lung Adenocarcinoma.
、 权利要求 1至 8任一项所述的疫苗组合物在治疗病毒感染、 细菌感染、 寄生 虫感染、 变态反应或癌症中的应用。 Use of the vaccine composition according to any one of claims 1 to 8 for the treatment of a viral infection, a bacterial infection, a parasitic infection, an allergy or cancer.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
WO2012160183A1 (en) * 2011-05-26 2012-11-29 Intervet International B.V. Immunostimulatory oligodeoxynucleotides
JP2014516534A (en) * 2011-05-26 2014-07-17 インターベット インターナショナル ベー． フェー． Immunostimulatory oligodeoxynucleotides
US20160158347A1 (en) * 2013-07-26 2016-06-09 Intervet Inc. Acceleration of Vector Virus Induced Immune Response in Avians
US9364531B2 (en) 2010-12-30 2016-06-14 Intervet Inc. Immunostimulatory oligodeoxynucleotides
WO2022162177A1 (en) * 2021-01-29 2022-08-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Chlamydia trachomatis antigenic polypeptides and uses thereof for vaccine purposes

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007012285A1 (en) * 2005-07-28 2007-02-01 Changchun Huapu Biotechnology Co., Ltd. Viral infection resistent single strand deoxynucleosides
CN101333237B (en) * 2007-06-26 2012-07-04 长春华普生物技术有限公司 Oligonucleotide with breast carcinoma treating function
CN109234280B (en) * 2018-11-02 2021-08-24 吉林农业大学 Sika deer specific CpG oligodeoxynucleotide and application thereof
CN114796476A (en) * 2021-09-24 2022-07-29 中国医学科学院医学生物学研究所 Novel nucleic acid adjuvant system of subunit vaccine and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1362263A (en) * 2001-01-04 2002-08-07 北京迪威华宇生物技术有限公司 Recombinant protein vaccine for preventing and treating human prostata cancer
WO2003025119A2 (en) * 2001-08-03 2003-03-27 Medarex, Inc. Compositions comprising immunostimulatory oligonucleotides and uses thereof to enhance fc receptor-mediated immunotherapies
CN1462636A (en) * 2002-05-30 2003-12-24 北京迪威华宇生物技术有限公司 Vaccine of recombined albumen for preventing and treating infection of human C type hepatitis virus and its usage
CN1478549A (en) * 2002-08-29 2004-03-03 \ Recombination protein for preventing human trachoma bedsnia infestation and its use
CN1526719A (en) * 2003-03-05 2004-09-08 长春华普生物技术有限公司 Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine
JP2004315433A (en) * 2003-04-16 2004-11-11 Woong-Shick Ahn PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING PAPILLOMA VIRUS-INDUCED DISEASE CONTAINING ANTIGENIC PROTEIN OF PAPILLOMA VIRUS AND CpG-OLIGODEOXYNUCLEOTIDE

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1362263A (en) * 2001-01-04 2002-08-07 北京迪威华宇生物技术有限公司 Recombinant protein vaccine for preventing and treating human prostata cancer
WO2003025119A2 (en) * 2001-08-03 2003-03-27 Medarex, Inc. Compositions comprising immunostimulatory oligonucleotides and uses thereof to enhance fc receptor-mediated immunotherapies
CN1462636A (en) * 2002-05-30 2003-12-24 北京迪威华宇生物技术有限公司 Vaccine of recombined albumen for preventing and treating infection of human C type hepatitis virus and its usage
CN1478549A (en) * 2002-08-29 2004-03-03 \ Recombination protein for preventing human trachoma bedsnia infestation and its use
CN1526719A (en) * 2003-03-05 2004-09-08 长春华普生物技术有限公司 Deoxyoligonucleotide containing CpG single strand for strengthening immunological effect of Protein vaccine
JP2004315433A (en) * 2003-04-16 2004-11-11 Woong-Shick Ahn PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING PAPILLOMA VIRUS-INDUCED DISEASE CONTAINING ANTIGENIC PROTEIN OF PAPILLOMA VIRUS AND CpG-OLIGODEOXYNUCLEOTIDE

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KLINMAN D.M. ET AL.: "CpG motifs as immune adjuvants", VACCINE, vol. 17, no. 1, January 1999 (1999-01-01), pages 19 - 25, XP004139028 *
LIPFORD G.B. ET AL.: "CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants", EUR. J. IMMUNOL., vol. 27, no. 9, September 1997 (1997-09-01), pages 2340 - 2344, XP002123073 *
MCCLUSKIE M.J. ET AL.: "CpG DNA is an effective oral adjuvant to protein antigens in mice", VACCINE, vol. 19, no. 7-8, 22 November 2000 (2000-11-22), pages 950 - 957, XP004225417 *

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WO2012160183A1 (en) * 2011-05-26 2012-11-29 Intervet International B.V. Immunostimulatory oligodeoxynucleotides
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