WO2008116380A1 - A vaccine preventing and/or treating autoimmune diseases - Google Patents

A vaccine preventing and/or treating autoimmune diseases Download PDF

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Publication number
WO2008116380A1
WO2008116380A1 PCT/CN2008/000540 CN2008000540W WO2008116380A1 WO 2008116380 A1 WO2008116380 A1 WO 2008116380A1 CN 2008000540 W CN2008000540 W CN 2008000540W WO 2008116380 A1 WO2008116380 A1 WO 2008116380A1
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protein
type
diabetes
antigen
cells
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PCT/CN2008/000540
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English (en)
French (fr)
Inventor
Bin Wang
Huali Jin
Youmin Kang
Wenjuan Zhang
Yang Yu
Jinyao Li
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China Agricultural University
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Priority to CA2681775A priority Critical patent/CA2681775C/en
Priority to JP2010500051A priority patent/JP5524823B2/ja
Priority to US12/532,643 priority patent/US20100143401A1/en
Priority to EP08714993.6A priority patent/EP2140884B1/en
Publication of WO2008116380A1 publication Critical patent/WO2008116380A1/zh
Priority to US13/706,948 priority patent/US20130089566A1/en

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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the present invention relates to a vaccine for preventing and/or treating autoimmune diseases.
  • An autoimmune disease is a disease caused by an autoimmune reaction of an autoantigen that causes damage to its own tissues.
  • Autoimmune diseases are common diseases such as type I diabetes, multiple sclerosis, rheumatoid arthritis, oophoritis, myocarditis, chronic thyroiditis, myasthenia gravis, lupus erythematosus, toxic diffuse goiter Graves' disease, dry synthesis Sign, uveal retinitis and so on.
  • the prevalence rate in China is over 5%.
  • the treatment of such diseases is mainly the use of immunosuppressive agents, and the immunosuppressive agents and methods commonly used in clinical settings are as follows:
  • the use of chemicals such as: Prograf
  • FK506 cyclosporine A (CsA), sputum (MMF), azathioprine (Aza), prednisone (Pred), early base prednisolone (MP); use of antibodies such as: anti-lymphocyte Protein (ALG), an anti-CD4 monoclonal antibody (0KT4).
  • ALG anti-lymphocyte Protein
  • KT4 anti-CD4 monoclonal antibody
  • Type I diabetes is a type of autoimmune disease characterized by destruction of insulin-producing cells in islets by infiltration of islets by CD4+ T cells, CD8+ T cells, and macrophages. It accounts for approximately 5-10% of all diabetic patients (ADA)
  • type I diabetes islet inflammation that is, lymphocytes infiltrate islets, and anti-islet cell autoantibodies (ICA) are found in patients with type I diabetes, and T, which reacts independently to insulin, carboxypeptidase, and heat shock proteins.
  • ICA anti-islet cell autoantibodies
  • Beakkeskov demonstrated that the 64K antibody present in the serum of patients with type 1 diabetes is glutamate decarboxylase (GAD) autoantibodies and autonomically reactive T cells, and GAD is considered to be a key antigen for autoimmune response in type I diabetes (Immune) Modulation for prevention of type 1 diabetes mellitus.
  • GAD glutamate decarboxylase
  • the vaccine for preventing and/or treating autoimmune diseases provided by the present invention, the active ingredient thereof is a mixture of: a protein antigen or an epitope polypeptide thereof which causes an autoimmune disease and a self-protein antigen or a table thereof inserted at a multiple cloning site a mixture of recombinant eukaryotic expression vectors of a polypeptide-encoding gene;
  • the self-protein antigen is insulin, glutamate decarboxylase, heat shock protein, myelin oligodendrocyte glycoprotein
  • M0G myelin antigens
  • MBP and PLP protein lipoprotein zona pellucida 3
  • ZP3 zona pellucida 3
  • myosin type II collagen, thyroglobulin, cell membrane surface antigen, type II glial antigen (CA2), acetylcholine receptor, thyroid cell surface antigen (TSH), salivary glandular protein, thyroglobulin, antigen including superantigen (S-Ag) or photoreceptor receptor resin-like binding protein.
  • S-Ag superantigen
  • S-Ag photoreceptor receptor resin-like binding protein.
  • the above vaccine for preventing and/or treating autoimmune diseases may specifically be a vaccine for preventing and/or treating type I diabetes.
  • the vaccine for preventing and/or treating type I diabetes the active ingredient of which is any one of the following:
  • the type I diabetes self-protein antigen is insulin or glutamate decarboxylase or heat shock protein.
  • a mixture of a type I diabetes self-protein antigen and a recombinant eukaryotic expression vector in which the type I diabetes self-protein epitope polypeptide-encoding gene is inserted at a multiple cloning site may also be used.
  • This mixture also produces regulatory T cells that inhibit the development of type 1 diabetes.
  • a mixture of a type I diabetes self-protein epitope polypeptide and a recombinant eukaryotic expression vector in which the type I diabetes autoantibody antigen-encoding gene is inserted at a multiple cloning site can also be used.
  • This mixture can also produce regulatory T cells to inhibit the development of type 1 diabetes.
  • the effect of immunization using the above two mixtures is produced by a mixture of a type I diabetes self-protein epitope polypeptide and a recombinant eukaryotic expression vector in which the type I diabetes self-protein epitope polypeptide is inserted at the multiple cloning site.
  • the effect is the same.
  • the insulin may be derived from a human, a dog or a cat.
  • Human insulin can be used in the treatment of type I diabetes in mice Treatment.
  • the genetic sequences of humans, dogs, cats, and mice are very similar. At the nucleic acid sequence level, the similarity between murine and human insulin is very similar.
  • the glutamic acid decarboxylase can be derived from humans, dogs, and cats. Human glutamate decarboxylase is also used in the treatment of type I diabetes in mice. At the nucleic acid sequence level, the sequence similarity of the two is 90%.
  • the heat shock protein can be derived from humans, dogs, and cats.
  • the type I diabetes self-protein antigen may specifically be human insulin.
  • the amino acid sequence of the type I diabetes self-protein epitope polypeptide is the sequence 1 in the sequence listing, and the polypeptide is named B9-23.
  • the eukaryotic expression vector for inserting the type I diabetes autoantibody antigen encoding gene or the type I diabetes autoantibody epitope polypeptide encoding gene may be a mammalian cell expression vector such as pcDNA3.0 or pVAXl or provax ( Tu Yizhen, Jin Huali, Zhang Xinyu, Yang Ruoye, Yang Fu, Zhang Fuchun, Wang Bin. Comparison of expression efficiency and immune effect of eukaryotic expression vector of classical swine fever virus E2 gene. Journal of China Agricultural University, 2005, 10 (6) : 37- 41).
  • the active ingredient of the vaccine for preventing and/or treating type I diabetes may specifically be human insulin protein and pVAX-insulin, and may also be B9-23 and pcDB9-23.
  • a type I diabetes self-protein antigen and a recombinant eukaryotic expression vector in which the type I diabetes autoantibody antigen-encoding gene is inserted at a multiple cloning site The mass ratio is 1: 5-5: 1; preferably 1: 1: 1: 2;
  • the mass ratio of the type I diabetes self-protein epitope polypeptide and the recombinant eukaryotic expression vector in which the type I diabetes self-protein epitope polypeptide is inserted at the multiple cloning site is 1: 5-5: 1; Preferably 1: 1 : 1 : 2;
  • the mass ratio of the type I diabetes self-protein antigen and the recombinant eukaryotic expression vector in which the type I diabetes autoantiprotein epitope polypeptide is inserted at the multiple cloning site is 1:5-5:1; preferably 1 : 1-1: 2;
  • the mass ratio of the type I diabetes self-protein epitope polypeptide and the recombinant eukaryotic expression vector in which the type I diabetes autoantibody antigen-encoding gene is inserted at the multiple cloning site is 1: 5- 5 : 1; preferably 1 : 1- 1: 2.
  • the vaccine for preventing and/or treating type I diabetes can be introduced into a body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, jetting, nasal drops, eye drops, permeation, absorption, physical or chemical mediated methods; Or be mixed or wrapped with other substances and then introduced into the body.
  • the vaccine for preventing and/or treating type I diabetes is generally used in an amount of 200 ug to 10 mg of active ingredient per kg of body weight per time, and is administered once every 7 to 30 days, generally 2 to 5 times.
  • this vaccine method and means can also be used for other autoimmune diseases induced by autoantigens.
  • Figure la shows the results of EcoR I and Xho I digestion of pVAX-insulin.
  • Figure lb shows the results of EcoR I and Xho I digestion of the pcDB9-23 vector.
  • Figure lc shows the expression of pVAX-insulin in BHK21 cells by RT-PCR.
  • Figure 2a shows the increase in T cells in the pcDB9-23 and B9-23 co-immunized groups stimulated for 48 hours.
  • Figure 2b shows the increase in T cells in the PCDB9-23 and B9-23 co-immunized groups stimulated for 96 hours.
  • Figure 3a shows the increase in T cells in each immunized group stimulated for 48 hours.
  • Figure 3b is a comparison of T cell proliferation in each of the immunized groups in Figure 3a.
  • Figure 3c is a comparison of T cell proliferation at different immunization doses in different co-immunization groups.
  • Figure 4 is a comparative experiment to prevent the onset of NOD mice.
  • Figure 5 shows the results of HE staining of pancreatic tissue sections of immunized NOD mice.
  • Figure 6a shows the nucleic acid source analysis of the mouse MZP3 gene sequence in the same manner as marmoset, human, dog, cat, Erhualian and Asian bovine.
  • MZP3 mouse; Marmosets: marmoset; Human: human; Canis familiaris: dog; felis catus: cat; Sus scrofa: Erhualian pig; Bos taurus: Asian original cattle.
  • Figure 6b shows the amino acid sequence homology analysis of the mouse MZP3 gene sequence with marmoset, human, dog, cat, Erhualian and Asian bovine.
  • MZP3 mouse; Marmosets: marmoset; Human: human; Canis familiaris: dog; felis catus: cat; Sus scrofa: Erhualian pig; Bos taurus: Asian original cattle.
  • Figure 7 shows the identification and expression analysis of the eukaryotic expression vector pcDmzp3.
  • the vector was digested with EcoR I and Xho I.
  • M DNA standard molecular weight (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and lOObp);
  • 1, 2 Recombinant plasmid pcDmzp3 was digested with EcoR I and Xho I.
  • M DNA standard molecular weight (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and lOObp); 1: PCDmzp3 transfected cells MZP3 expression; 2: Untransfected cell controls.
  • Figure 8 shows the identification, protein expression and purification of the prokaryotic expression vector pGEX-4T-1/MZP3.
  • M DNA standard molecular weight (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and lOObp);
  • 1, 2 The recombinant plasmid pGEX-4T-1/MZP3 was digested with EcoR I and Xho I.
  • M protein standard molecular weight
  • 1 whole bacterial protein induced by IPTG
  • 2 IPTG-induced whole bacterial protein
  • 3 sonicated supernatant protein
  • 4 sonicated protein.
  • M protein standard molecular weight
  • 1 purified protein protein sample. The arrow points to the destination strip.
  • Figure 9a shows the incidence and severity of autoimmune disease oophoritis.
  • C57BL/6 mice were injected through the soles of the feet and muscles.
  • lOOul PBS was used as a negative control group.
  • Only 100 ⁇ g of Freund's complete adjuvant (CFA) mice were used as an adjuvant group, and 100 ⁇ l of CFA emulsion containing 100 ⁇ g of MZP3 protein was injected.
  • CFA Freund's complete adjuvant
  • Figure % shows ovarian histological changes 14 days after injection.
  • C57BL/6 mice were injected with lOOul PBS as a negative control group through the soles of the feet and muscles. Only 100 ⁇ g of Freund's complete adjuvant (CFA) mice were used as an adjuvant group, and 100 ⁇ l of CFA emulsion containing 100 MZP3 protein was injected.
  • Negative control group normal ovarian tissue; CFA, mouse ovarian tissue injected only with CFA, no inflammatory response in the ovary; long arrow indicates growing follicles, arrows indicate primordial follicles (magnifications 100 and 400 times); MZP3, ovarian appearance Infiltration of inflammatory cells, severe loss of eggs.
  • Figure 9c shows antibody titers (3 mice per group).
  • C57BL/6 mice were injected with 100 ⁇ g of MZP3 protein and CFA through the soles of the feet and muscles, and the controls were mice injected only with CFA.
  • Serum was collected 14 days after the injection and the antibody was detected by ELISA, and the antibody titer was calculated based on the 0D value.
  • Figure 9 d is a specific T cell response to MZP3 protein from lymph nodes in the leg.
  • C57BL/6 Mice were injected with lOOul PBS as a negative control group through the soles of the feet and muscles, and only 10 ul of Freund's complete adjuvant (CFA) mice were used as an adjuvant group, and 100 ul of a CFA emulsion containing 100 ⁇ g of MZP3 protein was injected.
  • T cells were isolated from mice (3 in each group), stimulated with MZP3 protein as a specific antigen in vitro and detected by CFSE method.
  • the percentage in the figure is the degree of proliferation, and the larger the value indicates the higher the degree of proliferation, and Ml represents the percentage of cell proliferation.
  • Figure 10a shows the expression of the cytokine IL-2.
  • C57BL/6 mice were injected with lOOul PBS as a negative control group (Naive) through the soles of the feet and muscles. Only 100 ⁇ l of CFA mice were used as adjuvant groups, and 100 ⁇ l of CFA emulsion containing 100 ⁇ g of MZP3 protein was injected.
  • Figure 10b shows the analysis of IFN- ⁇ expression by flow cytometry.
  • T cells were isolated from the spleen and leg bone lymph nodes of C57BL/6 mice and stimulated with MZP3 protein for 8 hours in vitro.
  • the expression of INF- ⁇ in CD4+ cells and the expression of IL-12 in total cells were analyzed by intracellular staining. The percentage of positive cells is shown in the figure. a, lymph node; b, spleen.
  • Figure 10c shows the expression of IL-12 by flow cytometry.
  • T cells were isolated from the spleen and leg lymph nodes of C57BL/6 mice and stimulated with MZP3 protein for 8 hours in vitro.
  • the expression of INF- ⁇ in CD4+ cells and the expression of IL-12 in total cells were analyzed by intracellular staining. The percentage of positive cells is shown in the figure. a, lymph nodes; b, spleen.
  • Figure 11a shows that the co-immunized pcDmz P3 and MZP3 proteome A0D was alleviated compared to the control group.
  • Figure lib is the ovarian histology test on the 7th day after the second immunization
  • Figure 11c shows the expression of the cytokine IL-12 by flow cytometry.
  • A lymph nodes;
  • B spleen.
  • Figure l id is the expression of cytokine INF- Y detected by flow cytometry.
  • A lymph nodes; B, spleen.
  • Figure lie induces antigen-specific immunosuppression for co-immunization of DNA and proteins.
  • T cell expansion of antigen-matched and antigen-mismatched DNA and protein mice was co-immunized.
  • T cells were isolated on day 7 after the second immunization, re-stimulated in vitro with the specific antigen MZP3 protein, BSA as an unrelated protein control, and Con A as a positive control. Detection was performed using the MTT method and expressed as a stimulation index.
  • Figure 11f shows the detection of antibody levels in serum by ELISA.
  • Figure 12a shows the expression of IL-10 by regulatory T cells induced by co-immunization.
  • T cells were isolated on day 7 after the second immunization and analyzed for expression of cytokines IL-10 and FoxP3 by flow cytometry.
  • A lymph nodes; B, spleen.
  • Figure 12b shows the expression of FoxP3 by co-immunization-induced regulatory T cells.
  • T cells were isolated on day 7 after the second immunization and analyzed for expression of cytokines IL-10 and FoxP3 by flow cytometry.
  • A lymph nodes; B, spleen.
  • FIG. 13 shows that co-immunization did not alter the number of CD4+CD25+ cells.
  • T cells were isolated from mice immunized with different combinations, anti-CD4 and anti-CD25 monoclonal antibodies were stained and analyzed by flow cytometry.
  • A lymph nodes; B, spleen.
  • Figure 14 shows the results of restriction enzyme digestion of plasmid T-M0G352C.
  • Figure 15 shows the results of restriction enzyme digestion of plasmid pVAXM0G352c.
  • Figure 16 shows the results of transient expression of plasmid pVAXM0G352c in BHK21 cells.
  • Figure 17 shows the incidence of immune-induced mouse EAE model.
  • the ordinate is the incidence index and the abscissa is the number of days.
  • Fig. 18 shows the incidence of the M0G antigen emulsified by the CFA adjuvant after the subcutaneous immunization of the T cells derived from the spleen of the infected mouse, and the ordinate is the incidence index, and the abscissa is the number of days.
  • Figure 19 is a graph showing the incidence of lymph node-derived T cells in a negative-negative mouse after subcutaneous immunization with a CFA adjuvant emulsified M0G antigen for 200 ug. The ordinate is the incidence index and the abscissa is the number of days.
  • Figure 20 shows the expansion of T cells against their own M0G antigen after induction of mouse onset.
  • Figure 21 shows the expression of some cytokines inside T cells against their own M0G antigen after the onset of mice. The best way to implement the invention
  • Example 1 Vaccine for prevention and/or treatment of type I diabetes
  • vaccines for preventing and/or treating type I diabetes in this embodiment: 1) consisting of human insulin protein (Sigma, 1-9278) and pVAX-insulin, 2) 9-fold from the B chain of human insulin. 23 proteins (B9-23) and pcDB9-23, 3) composed of human insulin protein (Sigma, 1-9278) and pcDB9-23, 4) composed of B9-23 and pVAX-insulin.
  • the amino acid sequence of B9-23 is: S H L V E A L Y L V C G E R G (sequence 1).
  • the 9-23 protein (B9-23) on the B chain of insulin was synthesized by Beijing Aoke Company.
  • pVAX-insulin and pcDB9-23 were constructed as follows:
  • RNA extraction kit purchased from Takara Corporation, and total RNA was extracted according to the instructions. Primers were designed based on published gene sequences. Primer sequences are: Primers for amplifying human insulin cDNA:
  • the amino acid sequence of B9-23 is: SHLVEALYLVCGERG. ⁇ 'According to the TaKaRa RNA PGR Kit operating instructions, reverse transcription with Oligo (dT) as a downstream primer, reaction conditions: 42 ° C for 30 min, 99 ° C for 5 min, 5 ° C for 5 min. Specific primers were designed for the PCR reaction using the gene sequence. The amplification parameters were: 94 ° C for 2 min ; 94 ° C for 30 s, 55. C30s, 72°C, 70s, 35 cycles; 72°C for 10min, the reaction product was detected by 1% agarose gel electrophoresis. The results showed that the RT-PCR product of human insulin obtained a band of about 750 bp, and B9-23 RT-PCR. The product gave a band of approximately 250 bp.
  • the PCR product was recovered using Takara PCR Fragment Recovery Kit according to its instructions.
  • the recovered cDNA fragment was ligated with pMD 18-T vector (purchased from Takara) under the action of T 4 DNA ligase overnight at 16 ° C to ligate the cDNA encoding human insulin or the coding sequence of B9-23 to the vector of pMD 18-T. on.
  • Ligation of product-transformed competent cells, preparation and transformation of competent cells and plasmid extraction by reference (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, (2 nd ed) . New York : Cold Spring Harbor Laboratory Press, 1989. 19-56).
  • the extracted plasmid was digested with EcoR I and Hind III to identify the recombinant plasmid. After the digestion reaction, the agarose gel electrophoresis was carried out, and the correct clone was used to extract a large amount of the plasmid. To identify the cloned cDNA sequence, the recombinant plasmid was purified and BcaBEST primer RV-M and BcaBEST primer Ml 3-47 were used to convert the cDNA into the PE377 fully automated sequencer. The two-way DNA sequence was determined, and the resulting sequence was analyzed using PE SeqEd vl. 0.3 software.
  • the plasmid containing the human insulin cDNA gene of the GenBank Accession Number AY899304 from the 56th to the 388 deoxyribonucleotides of the 5' end of the GenBank Accession Number AY899304 was identified as pMD-insulin, containing nucleotides.
  • the plasmid having the sequence of the cDNA gene of B9-23 from the 5th end of the 5th end of the GenBank Accession Number AY899304 to the 196th deoxyribonucleotide was named pMD-B9-23.
  • the human insulin gene fragment and the B9-23 fragment were excised from pMD-insulin and pMD-B9-23, respectively, using Ec I and Iho I, and the human insulin gene fragment was ligated to the same eukaryotic expression vector pVAXl (Ir itrogen).
  • the B9-23 fragment was ligated into the same eukaryotic expression vector pcDNA3.1 (Ir itrogen), and the recombinant plasmid was identified by double restriction enzyme digestion and sequencing with EcoR I and Xho I.
  • the plasmid encoding the human insulin cDNA gene containing the human insulin deoxyribonucleotide from the 5' end to the 388 deoxyribonucleotide of the nucleotide sequence GenBank Accession Number AY899304 was identified as pVAX by restriction endonuclease digestion and sequencing.
  • the plasmid of the cDNA gene containing the nucleotide sequence of GenBank Accession Number AY899304 from the 5' end of position 152 to the 196 deoxyribonucleotide B9-23 was designated as pcDB9-23.
  • the pVAX-insulin reference lipofectaraine product specification was transfected with BHK21 cells (ATCC, USA) by lipofection. After transfecting the cells for 48 h, the cells were harvested and the total RNA was extracted with mlnsulinpl and mlnsulinp2 as primers to detect the expression of the target gene by RT-PCR. The results are shown in Figure lc, indicating that the 750 bp human insulin cDNA gene fragment in the transfected cells indicates that pVAX-insulin can be efficiently expressed in vitro at the mRNA level.
  • DNA standard molecular weight (10000 bp, 5000 bp, 2500 bp, lOOObp, and 250 bp, purchased from Takara); 2, 3: pVAX-insulin transfected cells insulin expression; 4: untransfected cell control.
  • Example 2 Effect test for prevention and/or treatment of type 1 diabetes vaccine
  • Balb/c mice and NOD mice for immunization experiments were separately used. Intramuscular injection. Balb/c mice were 3 in each group. N0D mice were 16 in each group.
  • mice Nine Balb/c mice were divided into 3 groups, 3 in each group.
  • the first group (pcDB9-23 immunized group) was each immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of pcDB9-23.
  • Group 2 (B9-23 immunized group) was each immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of B9-23, and the third group (pcDB9_23 and B9-23 co-immunized group) was each immunized with 100 Micrograms of pcDB9-23 and 100 micrograms of B9-23 in a 0.9% NaCl aqueous solution of 100 ⁇ l. The same dose was boosted once on the 14th day after the first immunization, and 7th after the second immunization.
  • the tau cell proliferation experiment was carried out as follows.
  • T cell proliferation assay T cell proliferation assay, CFSE staining, and flow cytometry were used to reflect the proliferative capacity of T lymphocytes against specific antigens. After receiving specific or non-specific stimulation of antigens, lymphocytes in the collection cause cell activation, cytokine synthesis, cytokine receptor expression and activation of cells to proliferate. The cell proliferation response can reflect the functional status of the cell to a certain extent.
  • mice were sacrificed by dislocation and soaked in 70% ethanol for 15 minutes. 2.
  • the spleen of the mice was removed under sterile conditions in an ultra-clean bench that was sterilized by ultraviolet light for 20 minutes in advance, and 2 ml of RPMI1640 was added in advance.
  • the cell culture dish 3 after the copper net is burned, cool it into a dish, grind the spleen with a sterile syringe, make a cell suspension, and filter into a 13 ml cell centrifuge tube; 4.
  • ⁇ Con A mitogen
  • B9-23 the corresponding specific antigen
  • Add one ⁇ BSA to a final concentration of 2 g/ml as an unrelated antigen.
  • the 48-hour and 96-hour test results showed that the degree of T cell proliferation (proliferation 0.08%) was significantly lower in the pcDB9-23 and B9-23 co-immunized groups than in the pcDB9-23 immunized group (22.32% proliferation) and B9- In the 23 immunized group (proliferation 8.94), it was confirmed that the pcDB9-23 and B9-23 co-immunization groups showed immunosuppression.
  • the percentages in Figures 2a and 2b are the degree of proliferation, the larger the value indicates the higher the degree of proliferation, and Ml represents the percentage of cell proliferation.
  • mice were divided into 7 groups, 3 in each group.
  • Group 1 negative control group
  • group 2 pVAX-insulin immunization
  • Each group was immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of pVAX-insulin, respectively.
  • the group of the three groups was each immunized with 100 ⁇ g of human insulin protein, respectively, with a solution of 100 ⁇ g of the human insulin protein, 100 ⁇ l, and the fourth group (pVAX1 immunized group) was each immunized with 100 ⁇ g of pVAX1.
  • 100 ⁇ L of % NaCl solution, group 5 (pVAX-insulin and VP1 co-immunization group) were each immunized with 100 ⁇ g pVAX-insulin and 100 ⁇ g foot-and-mouth disease virus VP1 (according to the literature: Jin Huali, Zhang Fuchun, Shan Wenjuan, Zhang Ailian , Li Jiejie, Wang Bin.
  • the percentage in Figure 3a is the degree of proliferation, with larger values indicating more proliferative levels and Ml indicating the percentage of cell proliferation.
  • pi represents the pVAX-insulin immunization group
  • Insulin represents the human insulin protein immunization group
  • pVAX represents the pVAX1 immunization group
  • pI+VP1 represents the pVAX-insulin and VP1 co-immunization group
  • pVAX+In represents pVAX1 and human insulin protein co-immunization.
  • pl + In indicates pVAX-insulin and human insulin protein co-immunization group
  • naive indicates negative control group.
  • mice were divided into 8 groups, 3 in each group.
  • the first group (negative control group) was each immunized with 0.9% NaCl aqueous solution 100 ⁇ l
  • the second group pVAX-insulin immunization
  • the 9% NaCl aqueous solution containing 100 micrograms of human insulin protein was each immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of pVAX-insulin, respectively.
  • group 4 pVAX-insulin and human insulin protein 1:4 co-immunization group each immunized with 0.5 ⁇ g of pVAX-insulin and 100 ⁇ g of human insulin protein in a 0.9% NaCl aqueous solution 100 ⁇ l
  • Group 5 pVAX-insulin and human insulin protein 1: 2 co-immunization group
  • Group 6 pVAX- Insulin and human insulin protein 1: 1 co-immunization group each immunized with 100 ⁇ g of pVAX- insulin and 100 ⁇ g of human insulin protein in a 0.9% NaCl aqueous solution 100 ⁇ l
  • group 7 each immunized with 100 ⁇ g of pVAX- insulin and 100 ⁇ g of human insulin protein in a 0.9% NaCl aqueous solution 100
  • pVAX-insulin and human insulin protein 2 1 co-immunized group
  • group 8 pVAX-insulin and The human insulin protein 4:1 co-immunized group was each immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 400 ⁇ g of pVAX-insulin and 100 ⁇ g of human insulin protein, respectively.
  • the T cell proliferation experiment was carried out by the method of the above step 1.
  • the results after 48 hours of stimulation are shown in Figure 3c.
  • the ratio between plasmid and protein is 2: 1, the inhibition is most pronounced.
  • na'ive indicates a negative control group
  • pi indicates a pVAX-insulin immunization group
  • In indicates a human insulin protein immunization group
  • 1:4, 1:2, 1:1, 2:1, 4:1 respectively represent pVAX - insulin and human insulin protein 1: 4 co-immunization group
  • pVAX-insulin and human insulin protein 1 1 co-immunization group
  • pVAX-insulin and human insulin protein 2 1 co-immunization group
  • pVAX-insulin and human insulin protein 4 1 co-immunization group.
  • NOD mice co-immunization prevention experiment 64 female NOD mice were divided into 4 groups, 16 in each group.
  • the first group negative control group
  • group 2 pVAX-insulin immunization group
  • Each of the cells was immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of pVAX-insulin
  • the third group human insulin protein immunized group was each immunized with 100 ⁇ g of human insulin protein.
  • pVAX-insulin and human insulin protein co-immunization groups Each was immunized with 100 ⁇ l of a 0.9% NaCl aqueous solution containing 100 ⁇ g of pVAX-insulin and 100 ⁇ g of human insulin protein, respectively. The same dose was boosted once on the 14th day after the first immunization, and the blood glucose changes of the mice were measured and recorded weekly using a blood glucose meter (purchased from Beijing Yicheng Co., Ltd.) after the second immunization. If the blood glucose level of the mouse exceeds 200 mg/dl for two consecutive weeks, the NOD is considered to have diabetes.
  • Naive NOD indicates a negative control mouse
  • Insulin indicates a human insulin protein immunized group
  • pVAX-insulin indicates a pVAX-insulin immunized group
  • Insulin+pVAX-insulin indicates pVAX-insulin and human insulin protein co-immunization.
  • Pancreatic tissues of negative control mice, diseased diabetic mice, and pVAX-insulin and human insulin protein co-immunized groups were sectioned and HE stained. The results also showed that islets in the pVAX-insulin and human insulin protein co-immunization groups. In the tissue, lymphocyte infiltration was slightly milder than in the other diseased groups, and was similar to normal uninfected NOD mice (negative control mice) (Fig. 5).
  • this experiment demonstrates a new approach to prevent type 1 diabetes. Further exploration of relevant mechanisms may lead to new approaches to the treatment of autoimmune diseases in humans and animals.
  • Example 3 Treatment/prevention of autonomous oophoritis by co-immunization
  • Autoimmune ovarian disease (A0D: autoimmune ovarian disease) is one of the causes of early human ovarian failure (P0F: premature ovarian failure).
  • A0D's own protein antigen is egg zona pellucida (ZP3).
  • the zona pellucida is a layer of glycoprotein-containing eosinophilic membrane secreted by oocytes and primary oocytes in the early stage of primary oocyte maturation, composed of ZP1, ZP2, and ZP3 [4] .
  • the zona pellucida has two important functions in the process of fertilization: one is to attach the sperm to the zona pellucida, and the other is to induce sperm acrosome reaction after the sperm is combined to make the sperm enter the egg [s] , where ZP3 is ZP A major glycoprotein, as a primary receptor for sperm, is more important in the process of sperm-egg binding and has been considered an ideal sterile vaccine [6] .
  • autoimmune ovarian disease is considered to be one of the main causes of POF [9_11] ; Rhim et al. established a new mouse experimental animal using a short peptide (330 to 342) of the foot and subcutaneous injection of MZP3. The model is used for the study of ovarian autoimmune diseases, and this model can mimic the early stage ovarian abortion in women [12] .
  • This autoimmune disease is mediated by CD4+ T cells, and Lou et al. found that the production of autoantibodies can alter the distribution of T cell-mediated inflammatory responses and lead to loss of target organ function units [13] .
  • regulatory T cells can negatively regulate the body's immune response to autoantigens, thereby protecting the body from autoimmune diseases.
  • Natural regulatory T cells are generally CD4+CD25+ double positive T cells, which express a functional transcription factor, FoxP3, which is an important cellular component of the normal immune system [ 14] . The presence of such cells allows researchers to use antigen-specific regulatory T cells to treat autoimmune diseases and allograft rejection [17] . Samy et al. use antigen-dependent
  • CD4+CD25+ double positive regulatory T cells control the development of autoimmune disease, ovarian inflammation [18 ].
  • Jin et al found that protein- and DNA co-immunization can inhibit antigen-specific cellular responses [19] .
  • This inhibitory function may be related to the induction of antigen-specific regulatory T cells, so this paper uses mice as animals.
  • Model using protein and DNA co-immunization to suppress cellular immune responses to explore the treatment of autoimmune diseases - ovarian inflammation and the corresponding mechanism.
  • the PMD18-T sequencing vector was purchased from Takara Corporation, and the Escherichia coli DH5 strain was our laboratory-preserved strain.
  • RNA extraction kit, PCR product recovery kit, DNA marker, restriction enzyme, exTaq enzyme, and RT-PCR and PCR primers were purchased from Takara; the sequencing kit was purchased from PE Company of the United States, and other reagents were of analytical grade. .
  • MZP3 PI AATGAATTCATGAATTCCCAGACTCTGTGGC
  • MZP3 P2 TTACTCGAGTTAAGTCCAGCCTTCCACAGTCT
  • the amplified fragment is the 63rd to 1143th base of the nucleic acid sequence of MZP3.
  • RNA PCR Kit operating instructions
  • reverse transcription was performed using Oligo (dT) as a downstream primer.
  • the reaction conditions were: 42 ° C for 30 min, 99 ° C for 5 min, and 5 ° C for 5 min.
  • PCR primers were used to design specific primers with mzp3 gene sequence.
  • the amplification parameters were: 94 ° C for 2 min; 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 70 s for 35 cycles; 72 ° C for 10 min, and the reaction products were detected by agarose gel electrophoresis.
  • the PCR product was recovered according to the Takara PCR Fragment Recovery Kit instructions.
  • the recovered mzp3 cDNA fragment was ligated with the P MD 18-T vector at 16 ° C overnight under the action of ⁇ 4 DNA ligase, and the mzp3 cDNA was ligated to the pMD 18-T vector.
  • the ligation product transforms competent cells, and the preparation and transformation of the competent state is according to the reference (Sambrook J et al. 1989) [2 ° ] .
  • a small amount of plasmid DNA was extracted according to the relevant reference (Sambrook J et al. 1989). The extracted plasmid was identified by double digestion with BamH I and Hind III, and the recombinant plasmid was identified by agarose gel electrophoresis.
  • Ec I and X w I were digested and ligated into the same digested eukaryotic expression vector pcDNA3, which was named pcDmzp3, and the correct recombinant plasmid was sequence-identified for eukaryotic transfection experiments.
  • the pcDmzp3 was transfected into BHK21 cells by lipofection. The specific steps are shown in the instructions. After transfecting the cells for 48 h, the cells were harvested and the total RNA was extracted with MZP3 P1 and MZP3 P2 as primers to detect the expression of the target gene by RT-PCR.
  • the recombinant plasmid pMD18_T/MZP3 was digested with Eco/PI and ho I, ligated into the same eukaryotic expression vector pGEX-4T-1, and the correct recombinant plasmid was sequenced for protein expression. Individual colonies were picked and inoculated into fresh LB (Am P + 50 mg/L) medium and incubated overnight at 37 ⁇ . The next day, the seeds were transferred to fresh LB (containing Amp + 50 mg/L) medium at a 1% inoculation amount. When A600 reached 0. 6 ⁇ 0. 8, it was expressed at 37 ° C for 5 h under the induction of 1.0 mraol/L IPTG.
  • the cells were collected by centrifugation with 1 mL of bacterial solution, washed once with double distilled water, suspended in the lxSDS sample addition solution, boiled in a boiling water bath for 10 min, centrifuged at 12 000 g for 2 min, and 15 ⁇ L of the supernatant was taken for 100 g/L SDS-PAGE and stained with Coomassie brilliant blue.
  • the induced C0 U BL21 (DE3) was collected by centrifugation and washed with PBS using a 1/20 original culture volume of cell lysate L1 (10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA and The bacterial pellet was resuspended in 200 mmol/L NaCl. After 15 min in an ice bath, the bacteria were sonicated until the bacteria solution was no longer viscous. Then, the supernatant was removed by centrifugation at 12 000 g for 10 min at 4 ° C, and the precipitate was collected to obtain the original MZP3 inclusion body protein.
  • the initial inclusion body protein was washed with L1 and 4 M urea, incubated at 70 ° C for 10 min, centrifuged at 4 ° C, 12 000 g for 5 min, and repeated 3 times.
  • the precipitate was removed by incubating for 10 min at 4 °C, 4 °C, 12 000 g for 5 min, and the supernatant was placed in a dialysis bag in L2 (10 mmol/L Tris-HCl (pH 8.
  • Dialysis was carried out for 8 h in L NaCl), followed by dialysis for 8 h in L2 containing 2 M urea, and the concentration of urea was gradually decreased until the urea was removed from the protein solution, and the protein content was determined by Bradford.
  • the 2 mg/ml protein was mixed with 1: 1 complete Freund's adjuvant (CFA: complete Freund's adjuvant).
  • CFA complete Freund's adjuvant
  • each mouse was immunized with 100 ⁇ l through the soles of the feet and muscles, and 100 proteins were immunized per mouse. CFA-only mice were used as controls. Blood was collected 14 days later, serum was collected, and the corresponding antibody titer was detected by indirect ELISA.
  • the purified GST-MZP3 fusion protein was used as a standard antigen, and the specific antibody level of ⁇ 3 protein in the serum of the mouse was detected by ELISA.
  • the protein was diluted to 5 ⁇ g/ml, and the 96-well microtiter plate was coated with 100 ⁇ l/well for 4 overnight; the coating solution was discarded 3 times with PBST, and 5% skim milk powder-PBST was blocked at 37 °C for 1 h; After washing the plate, add different dilutions of mouse serum for 2 h at 37 °C ; after washing, add 1: 1000 horseradish peroxidase-labeled goat anti-mouse secondary antibody (HRP-IgG), 50 ⁇ l/well, After incubating at 37 °C for 2 h, discard it; wash the plate, add 50 ⁇ L/well TMB room temperature to avoid color reaction for 30 min; 2 mol/L sulfuric acid to stop the reaction, and measure the value of OM50/650 with a microplate reader.
  • HRP-IgG horseradish peroxidase-labeled goat anti-mouse secondary antibody
  • ovarian disease Histological evaluation of ovarian disease: The ovaries were fixed in Bouin's fixative for 24 hours, embedded in paraffin, and then serially sectioned (5 ⁇ m) and stained with H. E (hematoxylin and eosin). Ovarian pathology was graded according to severity, inflammation occurred in 1 interstitial region; inflammatory response at multiple sites increased in 2 and 3 or granuloma between follicles and inside; 4 follicle disappearance and ovarian atresia.
  • Antigen-specific lymphocyte proliferative response The mice were sacrificed 14 days after immunization, the lymph nodes of the leg were removed aseptically, the lymph nodes were ground, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium; After the plate was counted, adjust the cell concentration to lxl07mL. Take 2 ⁇ 10 7 cells, centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in sterile PBS, add 1.5 ⁇ M ⁇ CFSE (1 mmol/ml), gently shake at 37 °C for 10 min, and add an equal volume of bovine serum to terminate.
  • the reaction was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded and washed three times with PBS. Finally, the cells were resuspended in 1 ml of medium, and 100 ⁇ M cells were added per empty, and ⁇ 3 (10 ug/ml) protein was used to stimulate T cell proliferation.
  • BSA (2 g/ml) was used as a non-specific antigen control
  • ConA (10 g/ml) was used as a positive control
  • proliferation was detected by flow cytometry.
  • the cytokines IL-12 and INF- y were detected by intracellular staining: the mice were sacrificed 14 days after immunization, the spleens were aseptically removed, the spleen tissues were ground, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded, using 1- 2 mL red blood cells. The cells were treated with lysate for 2 to 3 min, 6-12 mL of RPMI 1640 medium containing 4% serum was added, 2000 rpm, centrifugation for 5 min, the supernatant was discarded, the cells were resuspended in the medium, and the cell concentration was adjusted after counting with a hemocytometer. To 2xl07mL.
  • Lymph node single cell suspension was prepared as above, 2 ⁇ 10 ⁇ cells (100 ⁇ ⁇ ) were added per well, stimulated with 10 g/ml ⁇ 3 protein for 4-6 hours, and monencin was added to inhibit 1- 2 hours. Then, intracellular staining was performed, and the expression of cytokines was measured by flow cytometry. The specific process is as follows: (1) The mice were sacrificed 21 days after immunization, and the spleen single cell suspension was prepared.
  • the dosage is taken according to the reagent instructions), and the cell intracellular analyte is labeled; (7) The intracellular staining antibody is fully mixed with the cells, and reacted at room temperature for 30 minutes in the dark; (8) Add 2-3 mL of the washing solution, fully Mix, centrifuge at 500xg for 5 minutes, discard the supernatant, and resuspend the cells with 300 washes per tube; (9) Flow cytometry analysis.
  • RT-PCR was used to detect cytokine IL-2: According to TRIZOL Reagent (Gibco) instructions, 10 7 cells were taken from each group, lysed with 1 ml TRIzol Reagent, then added with 200 ⁇ l chloroform, and mixed, 12000 r/min, 4 °C. After centrifugation for 15 min, the upper aqueous phase was transferred to a new EP tube, 500 ⁇ l of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 min, 12000 r/min, centrifuged at 4 ° C for 10 min, and the supernatant was discarded. The RNA was precipitated with 75% ethanol.
  • the concentration of 2 mg/ml protein was mixed with CFA1:1, and each mouse was immunized by the soles of the feet and muscles after emulsification.
  • mice After 100 ⁇ l, 14 d, 100 ⁇ g of pcDmzp3+MZP3 was immunized by muscle, and pcDNA3, pcDNA3+ MZP3, pcDmzp3, pcDmzp3+0VA and MZP3 were used as control group. After 14 days, the immunization was boosted. The spleen cells of the mice were subjected to T cell expansion experiments by MTT method, MZP3 protein antigens were used to stimulate T cell proliferation, BSA was used as a non-specific antigen control, and ConA was used as a positive control. The specific process is as follows: The proliferation activity of mouse T cells in vitro was detected by MTT assay.
  • lymph nodes of the mouse leg were taken, the lymph nodes were ground, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in medium to prepare a single cell suspension to adjust the cell concentration. To 3xl07mL, the rest of the process is the same as above.
  • cytokines IL-2, IL-12, INFi, IL-10, FoxP3 and CD25 and ovarian inflammation were evaluated as above.
  • RNA and reverse transcription using oligo-deoxythymidine as a primer to obtain single-stranded cDNA The PCR primers were amplified to obtain a fragment of the MZP3 gene.
  • the RT-PCR product was identified by 1% agarose gel electrophoresis, and a band of about 1 200 bp was observed, which was consistent with our expectation.
  • the RT-PCR product was ligated to the cloning vector pMD18-T to construct the recombinant plasmid pMD18- ⁇ /MZP3.
  • the recombinant plasmid was digested with Bam AI and ⁇ III, and a DNA fragment of about 1200 bp was excised, which was the same size as the predicted ligation fragment.
  • the pMD18-T/MZP3 recombinant plasmid was subjected to bidirectional sequencing using BcaBESTprimer RV2M and BcaBEST primer Ml 3247, and the sequencing result showed that the homology with the published sequence was 99°/. .
  • the homology of the velvet, monkey, dog, cat, pig, and bovine MZP3 genes was analyzed by DNAMAN, and the homology reached 73% (Fig. 6a), and the amino acid sequence homology reached 71% (Fig. 6b).
  • Recombinant plasmid PMD18-T/MZP3 and eukaryotic expression vector pcDNA3 were digested with EcoR I and Xho I, recombined and purified, ligated, and the MZP3 gene was cloned into eukaryotic expression vector pcDNA3 to obtain recombinant vector pcDmzp3.
  • the recombinant plasmid was digested with EcoR I and Xho I, and showed a band at about 1200 bp by a 0.7% agarose gel electrophoresis (Fig. 7 a), indicating that the recombinant plasmid pcDmzp3 was successfully constructed.
  • the purified and quantified plasmid was transfected into BHK21 cells with good growth condition by liposome, and the cells were collected at 72 h.
  • the expression of the target gene was detected by RT-PCR with MZP3pl and MZP3p2 as primers, and correspondingly appeared at 1200 bp.
  • the band is shown as 13 in Figure 7, indicating that the recombinant vector is efficiently expressed in vitro at the mRNA level.
  • the recombinant plasmid PMD18-T/MZP3 and the eukaryotic expression vector pGEX4T-1 were digested with EcoR I and Xho I, and then ligated and purified.
  • the MZP3 gene was cloned into the eukaryotic expression vector PGEX4T-1 to obtain the recombinant vector pGEX4T- 1/MZP3.
  • the recombinant plasmid was digested with EcoR I and Xho I, and showed a band at about 1200 bp by a 0.7% agarose gel electrophoresis (see a in Figure 8), indicating that the recombinant plasmid pGEX4T-l/MZP3 was successfully constructed.
  • the purified and quantified plasmid was transformed into competent cells ⁇ : coii BL2KDE3), and the constructed £ coii BL21 (DE3) / MZP3 transformants were cultured in fresh LB medium, and transformed bacteria were collected after induction by IPTG. Take the whole bacterial protein for SDS-PAGE.
  • Protein electrophoresis results showed that the MZP3 gene was expressed in a large amount in £ ⁇ 8121 (0 £ 3 ) ( b in Figure 8 ).
  • the cells after induction of expression were collected and treated with sonication.
  • SDS-PAGE results indicated that the induced expression of the protein band was present in the precipitate of the bacterial lysate, indicating that the protein was expressed as an inclusion body.
  • the protein was solubilized, washed and renatured by purifying the inclusion body to obtain a protein of higher purity (c in Figure 8).
  • the purified MZP3 protein was adjusted to a concentration of 2 mg/ml and an equal volume of complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • mice immunized with MZP3 protein and CFA developed varying degrees of ovarian inflammation with an incidence of 87.5% (Fig. 9a).
  • An inflammatory response is found in the interstitial of the ovary and in the growing and mature follicles. Follicles are infiltrated by inflammatory cells and severely cause loss of eggs (Fig. 9b).
  • mice injected with CFA did not develop ovarian inflammation.
  • mice were injected with MZP3 protein and CFA through the soles of the feet and muscles. After 14 days, the muscles were immunized with MZP3 protein and plasmid pcDmzp3, and boosted two weeks later. The incidence of ovarian inflammation was detected 7 days after the second immunization. As a result, it was found that only mice in which the co-immunization plasmids pcDmzp3 and MZP3 proteins (designated pcDm Z p3+MZP3) exhibited A0D inhibition (Fig. 11a).
  • Histological analysis also revealed inflammatory cell infiltration in the ovary of mice immunized with PCDNA3+MZP3 or pcDmzp3+0VA, whereas ovarian cells immunized with pcDmzp3+MZP3 did not have inflammatory cell infiltration (Fig. lib).
  • mice that have a total of pcDmz P 3+MZP3 The expression of IL-12 and INF- ⁇ in mice immunized with pcDNA3+MZP3 or pcDmz P 3+0VA was not inhibited, whereas mice immunized with pcDmzp3+MZP3 IL-12 (Fig. 11c) and INF- ⁇ (Fig. lid) Expression is suppressed. This suggests that mice that have a total of pcDmzp3+MZP3 have anti-inflammatory immune regulation.
  • T cells isolated from mouse spleens which were first immunized with MZP3 protein and CFA, and after 14 days immunized with pcDNA3, pcDNA3+MZP3, pcDmzp3, pcDmzp3+MZP3 , pcDmzp3+0VA and MZP3, boosted immunization two weeks later, and the spleen was removed on the 7th day after the second immunization.
  • T cells were used to analyze the ability to respond to MZP3 protein antigens.
  • T cells isolated from mice immunized with pcDmzp3+MZP3 showed substantially no amplification ability, while T cells isolated from other groups showed strong amplification ability (Fig. He).
  • MZP3 autonomic antibodies play an important role in guiding the distribution of autoimmune inflammatory responses and can enhance the severity of autoimmune diseases [13] . So we next tested whether co-immunization inhibited the production of anti-MZP3 antibodies. However, the results showed that the titer of the autonomic antibody was identical between the immunological groups pcDmzp3+MZP3, pcDNA3+MZP3 or MZP3 (Fig. 1 lf). This result is consistent with previous studies. If there is no T cell response, only transfer of antibodies cannot induce autoimmune ovarian disease [13] .
  • cytokines IL-10 and FoxP3 play important roles in the inhibition of T cell responses [27-29 ] .
  • T cells In order to detect induction by co-immunization Whether regulatory T cells can express characteristic cytokines and markers, we isolated T cells from mice immunized with pcDNA3, pcDNA3+MZP3, pcDmzp3 pcDmzp3+MZP3, pcDmzp3+0VA and MZP3, respectively. These cytokines or markers were intracellularly stained with specific fluorescently labeled antibodies and analyzed by flow cytometry.
  • CD4+CD25-regulatory T cells which suppress antigen-specific T cell responses and prevent autoimmune diseases.
  • This regulatory T cell has a phenotype of CD4+CD25-Foxp3 + , expresses IL-10 and is able to inhibit antigen-specific T cell responses and reduce the expression of cytokines IL-12 and INF- y.
  • Egg zona pellucida is very conserved in mammals. Establishing an A0D model in mice can better understand the pathogenesis of ovarian inflammation in other animals, especially human autoimmune diseases. Autoimmune disease oophoritis was induced in B6AF1 mice [(C57BL/6 XA/J) F1] by subcutaneous and subcutaneous injection of a 15-amino acid ZPP3 short peptide (328-342) with CFA [12 ' 13] . There was no ovarian inflammation in C57BL/6 mice. By injecting MZP3 protein and CFA in the soles of the feet and muscle, we successfully established an ovarian model on C57BL/6 mice, although the severity of the disease was not as previously reported [13] .
  • the MZP3 protein we use contains more T cell epitopes, which cause a stronger T cell immune response, and A0D is caused by T cell responses [12] .
  • A0D is caused by T cell responses [12] .
  • the natural regulatory T cell CD4+CD25+ can inhibit T cell responses and maintain autoimmune tolerance. If the regulatory T cells are removed, it can cause disease [32 ' 33 ' u so these cells play an important role in preventing the occurrence of autoimmune diseases. These characteristics make regulatory T cells a potential therapeutic tool for autoimmune diseases [35] . Recently, some studies have shown that regulatory T cells can indeed treat autoimmune diseases. These regulatory T cells can be antigen-unspecific [ 36-38] or antigen-specific [ 18'39] . Previously, we also reported that co-immunization of pcD-VP1 and 146S antigens induced T cell response immunosuppression [19] .
  • Antibodies to interleukin 12 abrogate established experimental colitis in mice.
  • Interleukin 12 protects from a T helper type 1 -mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon ⁇ , nitric oxide, and apoptosis. J. Exp. Med.
  • MS Multiple sclerosis
  • EAE Experimental allergic encephalomyelitis
  • the purpose of this experiment is to treat or prevent the occurrence of EAE in the EAE model of mice based on immunosuppression induced by co-immunization, and to further explore the related immune mechanisms of MS in the course of research.
  • the multiple protein sclerosis (MS) self-protein antigens are myelin oligodendrocyte glycoprotein (M0G) and two myelin antigens (MBP and PLP protein lipoprotein).
  • the PMD18-T sequencing vector was purchased from Takara Co., Ltd., and the E. coli DH5 ⁇ strain was our laboratory-preserved strain.
  • the PCR product recovery kit, DNA marker, restriction endonuclease, exTaq enzyme, and RT-PCR and PCR primers were purchased from Takara Corporation.
  • the sequencing kit was purchased from PE Company of the United States, and other reagents were of analytical grade.
  • the autoantigen commonly used in EAE models of induced animals is M0G (Myelin Oligodendrocyte
  • Glycoprotein 35-55 amino acids (amino acid sequence: MEVGWYRSPFSRVVHLYRNGK), its DNA sequence is: GAGGTGGGTTGGTACCGTTCTCCCTTCTCAAGAGTGGTTCACCTCTACCGAAATGGCAAG
  • the reaction product was detected by agarose gel electrophoresis.
  • the PCR product was recovered according to the Takara PCR Fragment Recovery Kit instructions.
  • the recovered M0G35-55 2copies fragment was ligated with the pMD18-T vector at 16 ° C overnight under the action of T4 DNA ligase, and ligated to the pMD 18-T vector.
  • the constructed plasmid was named as: T-M0G352C ligation product transformed into competent cells of DH5 ct strain.
  • the plasmid was extracted in small amounts and the recombinant plasmid was identified by double digestion with EcoRI I and Hind III. After the digestion reaction, the agarose gel electrophoresis was performed. To identify the cloned DNA sequence, the recombinant plasmid was purified and further sequenced, and the results were analyzed using DNAMAN software.
  • the plasmid with the correct sequence analysis was ligated to the same digested eukaryotic expression vector pVAX1 by BamH I and Hind III double-knife, and the vector was named pVAXM0G352c, and the correct recombinant plasmid was sequence-identified for eukaryotic transfection experiments.
  • pVAXM0G352c was transfected into BHK21 cells by lipofection. After transfecting the cells for 48 h, the cells were harvested and the total RNA was extracted with M0G35 P1 and M0G35 P2 as primers to detect the expression of the target gene by RT-PCR.
  • Fig. 14 The results of restriction enzyme digestion of plasmid T-M0G352c are shown in Fig. 14.
  • the results of restriction enzyme digestion of plasmid pVAXMOG352c are shown in Fig. 15.
  • the transient expression results of plasmid pVAXM0G352c in BHK21 cells are shown in Fig. 16.
  • the animal's tail is weak and soft.
  • the animal's tail is weak, and the forelimb or hind limb is moderately weak.
  • Figure 19 shows the lymph node-derived T cells of the negative mice after transfer to the mice, and the M0G antigen emulsified by the CFA adjuvant in the subcutaneous immunization 200 ug;
  • Figure 20 shows the expansion of sputum cells against their own M0G antigen after induction of mouse onset.
  • the C57 mouse was used to induce the sputum model, and the clinical symptoms were recorded until the clinical index of all mice reached 3 or higher.
  • the diseased mice were grouped as follows, and immunized separately, and the immunization method was intramuscular injection.
  • cytokines The expression of cytokines in spleen cells and the expression of cytokines in cerebrospinal fluid.
  • the next step is to perform immunological experiments to verify the application of co-immunization based on the establishment of a sound animal model.

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Description

一种预防和 /或治疗自身免疫疾病的疫苗 技术领域
本发明涉及一种预防和 /或治疗自身免疫疾病的疫苗。
背景技术
自身免疫疾病是由自身抗原发生免疫反应而导致自身组织损害所引起的疾病。 自身 免疫疾病是常见病，如 I型糖尿病、多发性硬化症、类风湿性关节炎、卵巢炎、心肌炎、 慢性甲状腺炎、重症肌无力、 红斑狼疮、毒性弥漫性甲状腺肿 Graves'病、干燥综合征、 葡萄膜视网膜炎等。 我国患病率超过 5%。 治疗此类疾病主要是使用免疫抑制剂， 在临 床上通常使用的免疫抑制剂和方法有如下几个方面： 利用化学药品如： 普乐可复
(FK506) , 环孢素 A(CsA)， 骁悉 (MMF)， 硫唑嘌呤 (Aza)， 强的松 (Pred)， 早基强的松龙 (MP) ; 利用抗体如： 抗淋巴细胞球蛋白（ALG) , 抗 CD4单克隆抗体 (0KT4)。 每年治疗费 用在几十亿元。但以上的免疫抑制剂都有其毒副作用， 若使用不当， 一方面可因过度抑 制机体免疫反应性而引发多种并发症，另一方面也可因其自身的毒副作用导致抑制器官 功能衰竭。 所以， 目前无特效的治疗手段。
I型糖尿病以 CD4+T细胞， CD8+T细胞和巨噬细胞浸润胰岛而造成胰岛中产生胰岛素 的细胞被破坏为特征的一类自身免疫疾病。 它约占所有糖尿病患者的 5- 10% (ADA
[American Diabetes Association] . 1997. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care
20： 1183-1197； Atkinson MA, Le iter EH. 1999. The NOD mouse model of type 1 diabetes： As good as it gets? Nature 5 : 601-604) 。 主要发病机理是自身反应的 T淋巴细胞破 坏了胰腺中产生胰岛素的细胞引起的， 以 CD4+ T细胞， CD8+ T细胞和巨噬细胞浸润胰岛 而造成胰岛中产生胰岛素的细胞被破坏为特征的一类自身免疫疾病 (Atkinson MA, Maclaren NK. 1994. The pathogenesis of insulin-dependent diabetes mellitus. N Engl J Med 331 : 1428-1436 ; Benoist C, Mathis D. 1997. Autoimmune diabetes : Retrovirus as trigger, precipitator or marker? Nature 388： 833-834； B jork S. 2001. The cost of diabetes and diabetes care. Diabetes Res Clin Pract 54 (Suppl 1) : 13-18) 0 在欧洲后裔中发病率较高， 约有 200万患者。 具有发病率明显的地理分布特 点: 芬兰儿童中 I型糖尿病的发病率是委内瑞拉儿童的 400倍。 有报道称， I型糖尿病 的全球发病率在 2010年将比在 1998年提髙 40°/。。 这个快速的增长速度表明， 环境因素作 用于易患基因， 共同引起了 1型糖尿病的发病率上升。 ，
人们已经发现 I型糖尿病胰岛炎现象， 即淋巴细胞浸润胰岛， 以后相继在 I型糖尿 病患者中发现了抗胰岛细胞自身抗体 (ICA)， 对胰岛素、 羧肽酶、 热休克蛋白产生自主 反应的 T细胞， 至 1990年 Beakkeskov证明 I型糖尿病患者血清中存在的 64 K抗体就 是谷氨酸脱羧酶 (GAD)自身抗体和自主反应的 T细胞， 认为 GAD是 I型糖尿病自身免疫 反应的关键抗原 (Immune modulation for prevention of type 1 diabetes mellitus. Itamar Razl, Roy El dor 2 and Yaakov Naparstek. TRENDS in Biotechnology 23: 128， 2005.龙秀荣， 杜文斌， 苏钟浦， 魏庆铮.儿童糖尿病的谷氨酸脱羧酶抗体检测. 中华儿科杂志. 1998年第 10期）。
目前临床上主要是给与胰岛素的补偿性治疗，但是还没有能够预防或延缓发病的好 方法。 围绕 1型糖尿病的研究方向有很多， 主要目标是抵抗自身免疫的发生， 到晚期也 有采取诱导细胞再生等方法。
发明公开
本发明的目的是提供一种预防和 /或治疗自身免疫疾病的疫苗。
本发明所提供的预防和 /或治疗自身免疫疾病的疫苗，它的活性成分是下述混合物: 造成自身免疫疾病的蛋白抗原或其表位多肽和在多克隆位点插入自身蛋白抗原或其表 位多肽编码基因的重组真核细胞表达载体组成的混合物；
所述自身蛋白抗原为胰岛素、 谷氨酸脱羧酶、 热休克蛋白、 髓磷脂寡突细胞糖蛋白
(M0G)、 二种髓磷脂抗原 (MBP和 PLP蛋白脂蛋白）、 卵透明带蛋白 3 (ZP3) 、 肌球蛋白、 II型胶原蛋白、 甲状腺球蛋白、 细胞膜表面抗原、 第二型胶质抗原 （CA2) 、 乙酰胆碱 受体、甲状腺细胞表面抗原（TSH)、唾液腺管蛋白、甲状腺球蛋白、抗原包括超抗原 (S - Ag) 或感光器间受体树脂样结合蛋白。
上述预防和 /或治疗自身免疫疾病的疫苗具体可为预防和 /或治疗 I型糖尿病的疫 苗。
所述预防和 /或治疗 I型糖尿病的疫苗， 它的活性成分是下述任一种混合物：
1 ) I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原编 码基因的重组真核细胞表达载体组成的混合物；
2) I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身蛋 白抗原表位多肽编码基因的重组真核细胞表达载体组成的混合物；
3) I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原表 位多肽编码基因的重组真核细胞表达载体组成的混合物；
4) I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身蛋 白抗原编码基因的重组真核细胞表达载体组成的混合物；
所述 I型糖尿病自身蛋白抗原为胰岛素或谷氨酸脱羧酶或热休克蛋白。
在共免疫组合物中，也可以釆用 I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原表位多肽编码基因的重组真核细胞表达载体组成的混合物。此 混合物同样可以产生调节性 T细胞而抑制 I型糖尿病发生。
同样，也可以采用 I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型 糖尿病自身蛋白抗原编码基因的重组真核细胞表达载体组成的混合物。此混合物同样可 以产生调节性 T细胞而抑制 I型糖尿病发生。
采用以上两种混合物免疫产生的效果与 I型糖尿病自身蛋白抗原表位多肽和在多 克隆位点插入所述 I型糖尿病自身蛋白抗原表位多肽编码基因的重组真核细胞表达载 体组成的混合物产生的效果相同。
其中， 所述胰岛素可来源于人、 狗、 猫。 人的胰岛素可以用于小鼠的 I型糖尿病治 疗。 人、 狗、 猫和鼠的基因序列非常相似。 在核酸序列水平上， 鼠与人胰岛素相似性为
95%, 猫与人的相似性为 84%， 狗与人的相似性为 89%。
所述谷氨酸脱羧酶可来源于人、 狗、猫。 人的谷氨酸脱羧酶也用于小鼠 I型糖尿病 治疗。 在核酸序列水平上， 两者的序列相似度为 90%。
所述热休克蛋白可来源于人、 狗、 猫。
所述 I型糖尿病自身蛋白抗原具体可为人胰岛素。所述 I型糖尿病自身蛋白抗原表 位多肽的氨基酸序列是序列表中的序列 1， 该多肽的名称为 B9- 23。 用于插入所述 I型 糖尿病自身蛋白抗原编码基因或所述 I型糖尿病自身蛋白抗原表位多肽编码基因的真 核细胞表达载体可为哺乳动物细胞表达载体，如 pcDNA3. 0或 pVAXl或 provax (涂亦娴， 金华利， 张馨玉， 杨若耶， 杨富， 张富春， 王宾。 猪瘟病毒 E2基因真核表达载体表达 效率和免疫效果的比较。 中国农业大学学报， 2005， 10 (6) ： 37-41 ) 。
所述预防和 /或治疗 I型糖尿病的疫苗的活性成分具体可为人胰岛素蛋白和 pVAX- insulin, 还可为 B9- 23和 pcDB9- 23。
所述预防和 /或治疗 I型糖尿病的疫苗的活性成分中， 1 ) I型糖尿病自身蛋白抗原 和在多克隆位点插入所述 I型糖尿病自身蛋白抗原编码基因的重组真核细胞表达载体 的质量比为 1 : 5-5 : 1; 优选为 1 : 1- 1: 2；
2) I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身蛋 白抗原表位多肽编码基因的重组真核细胞表达载体的质量比为 1 : 5-5 : 1;优选为 1 : 1- 1 : 2；
3) I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原表 位多肽编码基因的重组真核细胞表达载体的质量比为 1 : 5-5 : 1 ; 优选为 1 : 1-1: 2；
4) I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身蛋 白抗原编码基因的重组真核细胞表达载体的质量比为 1 : 5- 5 : 1; 优选为 1 : 1- 1: 2。
所述预防和 /或治疗 I型糖尿病的疫苗可通过注射、 喷射、 滴鼻、 滴眼、 渗透、 吸 收、 物理或化学介导的方法导入机体如肌肉、 皮内、 皮下、 静脉、 粘膜组织； 或是被其 他物质混合或包裹后导入机体。
所述预防和 /或治疗 I型糖尿病的疫苗的用量一般为 200ug— 10mg活性成分 /kg体 重 /次， 每 7— 30天给药一次， 一般共需 2- 5次。
小鼠实验证明， I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身 蛋白抗原编码基因的重组真核细胞表达载体组成的混合物，和 I型糖尿病自身蛋白抗原 表位多肽和在多克隆位点插入所述 I型糖尿病自身蛋白抗原表位多肽编码基因的重组 真核细胞表达载体组成的混合物，可抑制免疫小鼠的 τ细胞增殖，诱导免疫抑制的产生， 并能够有效地预防 I型糖尿病的发生。 同理， 此疫苗方法和手段也可以用于对于其它由 于自身抗原诱发的自身免疫性疾病。
附图说明
图 la为 pVAX- insulin的 EcoR I和 Xho I酶切鉴定结果。
图 lb为 pcDB9- 23载体的 EcoR I和 Xho I酶切鉴定结果。 图 lc为 RT- PCR方法分析 pVAX- insulin在 BHK21细胞中的表达。
图 2a为刺激 48小时的 pcDB9- 23和 B9-23共免疫组的 T细胞增情况。
图 2b为刺激 96小时的 PCDB9- 23和 B9-23共免疫组的 T细胞增情况。
图 3a为刺激 48小时的各免疫组的 T细胞增情况
图 3b为在图 3a中各免疫组的 T细胞增情况比较。
图 3c为在不同共免疫组中不同免疫剂量的 T细胞增情况比较。
图 4为预防 NOD小鼠发病的比较实验。
图 5为免疫的 NOD小鼠的胰腺组织切片 HE染色结果。
图 6a为小鼠 MZP3基因序列与绒猴、 人、 狗、 猫、 二花脸猪和亚洲原牛同的核酸源 性分析。 MZP3:小鼠； Marmosets:绒猴； Human:人； Canis familiaris:狗； felis catus: 猫； Sus scrofa: 二花脸猪； Bos taurus: 亚洲原牛。
图 6b为小鼠 MZP3基因序列与绒猴、 人、 狗、 猫、 二花脸猪和亚洲原牛的氨基酸序 列同源性分析。 MZP3: 小鼠； Marmosets: 绒猴； Human: 人； Canis familiaris: 狗； felis catus： 猫； Sus scrofa: 二花脸猪； Bos taurus： 亚洲原牛。
图 7为真核表达载体 pcDmzp3的鉴定及表达分析。 （a) 载体用 EcoR I和 Xho I进 行酶切分析。 M: DNA标准分子量（2000bp、 1000bp、 750bp、 500bp、 250bp和 lOObp) ； 1 , 2： 重组质粒 pcDmzp3用 EcoR I和 Xho I进行酶切。 （b) RT-PCR方法分析 pcDmzp3 在 BHK21细胞中的表达。 M: DNA标准分子量 (2000bp、 1000bp、 750bp、 500bp、 250bp 和 lOObp) ； 1： pcDmzp3转染的细胞 MZP3的表达； 2: 未转染的细胞对照。
图 8为原核表达载体 pGEX- 4T- 1/MZP3的鉴定， 蛋白表达及纯化。 （a)重组质粒 pGEX- 4T- 1/MZP3的酶切鉴定。 M: DNA标准分子量 （2000bp、 1000bp、 750bp、 500bp、 250bp和 lOObp) ； 1， 2： 重组质粒 pGEX- 4T- 1/MZP3用 EcoR I和 Xho I进行酶切。 （b) 蛋白的诱导表达。 M: 蛋白标准分子量； 1 : 未经 IPTG诱导的全菌蛋白； 2: IPTG诱导的 全菌蛋白； 3: 超声破碎的上清蛋白； 4: 超声破碎的沉淀蛋白。 （c)纯化后的 MZP3蛋 白。 M: 蛋白标准分子量； 1 : 纯化后的蛋白蛋白样品。 箭头指向目的条带。
图 9a为自身免疫疾病卵巢炎的发病率和严重程度。 C57BL/6小鼠通过脚掌和肌肉注 射 lOOul PBS为阴性对照组，只注射 lOOul弗氏完全佐剂 (CFA)的小鼠为佐剂组， 注射 lOOul的含 100 μ g MZP3蛋白的 CFA乳化液。
图％为注射后 14天检测卵巢组织学变化。 C57BL/6小鼠通过脚掌和肌肉注射 lOOul PBS为阴性对照组,只注射 lOOul弗氏完全佐剂 (CFA)的小鼠为佐剂组， 注射 lOOul的含 100 MZP3蛋白的 CFA乳化液。 阴性对照组， 正常卵巢组织； CFA， 只注射 CFA的小 鼠卵巢组织， 卵巢没有炎症反应；长箭头表示正在生长的卵泡，箭头表示原始卵泡 （放 大倍数为 100和 400倍） ； MZP3, 卵巢出现了炎症细胞的浸润， 严重的丢失了卵子。
图 9c为抗体滴度（每组 3只小鼠）。 C57BL/6小鼠通过脚掌和肌肉注射 100 μ g MZP3 蛋白和 CFA,对照为只注射 CFA的小鼠。注射 14天后收集血清并通过 ELISA的方法检测 抗体， 抗体滴度根据 0D值进行计算。
图 9 d为来自腿弯处淋巴结淋巴细胞对 MZP3蛋白的特异性 T细胞反应。 C57BL/6 小鼠通过脚掌和肌肉注射 lOOul PBS为阴性对照组，只注射 lOOul弗氏完全佐剂 (CFA)的 小鼠为佐剂组， 注射 lOOul的含 100 μ g MZP3蛋白的 CFA乳化液。 T细胞从小鼠中分离 (每组 3只） ， 体外用 MZP3蛋白作为特异性抗原进行刺激并用 CFSE方法进行检测。 图 中的百分数为增殖程度， 数值越大表明增殖程度越高， Ml表示细胞增值的百分比。
图 10a为细胞因子 IL- 2的表达。 C57BL/6小鼠通过脚掌和肌肉注射 lOOul PBS为阴 性对照组 (Naive) ,只注射 lOOul CFA的小鼠为佐剂组， 注射 lOOul的含 100 μ g MZP3 蛋白的 CFA乳化液。
图 10b为用流式细胞仪分析 IFN- γ 的表达。 注射 14天后， T细胞从 C57BL/6小鼠 的脾脏和腿弯处淋巴结分离并用 MZP3蛋白在体外刺激 8小时。 用胞内染色的方法分析 CD4+细胞 INF- γ的表达和总细胞 IL-12的表达。 阳性细胞的百分率显示在图中。 a， 淋 巴节； b， 脾脏。
图 10c为用流式细胞仪分的 IL- 12的表达。注射 14天后， T细胞从 C57BL/6小鼠的 脾脏和腿弯处淋巴结分离并用 MZP3蛋白在体外刺激 8小时。用胞内染色的方法分析 CD4+ 细胞 INF- γ 的表达和总细胞 IL- 12的表达。阳性细胞的百分率显示在图中。 a，淋巴节； b， 脾脏。
图 11a为和对照组相比， 共免疫 pcDmzP3和 MZP3 蛋白组 A0D被减轻了。
图 lib为第二次免疫后第 7天的卵巢组织学检测
图 11c为用流式细胞仪检测细胞因子 IL-12的表达。 A， 淋巴结； B， 脾脏。
图 l id为用流式细胞仪检测细胞因子 INF- Y 的表达。 A， 淋巴结； B, 脾脏。
图 lie为共免疫 DNA和蛋白诱导了抗原特异性的免疫抑制。比较共免疫了抗原匹配 型和抗原错配型 DNA和蛋白小鼠的 T细胞扩增情况。 第二次免疫后第 7天分离 T细胞， 体外用特异性抗原 MZP3蛋白重新刺激， BSA作为无关蛋白对照， Con A作为阳性对照。 用 MTT方法进行检测并以刺激指数的方式表示。 - ''
图 llf为用 ELISA法检测血清中的抗体水平。
图 12a为共免疫诱导的调节性 T细胞表达 IL- 10。 第二次免疫后第 7天分离 T细胞 并用流式细胞仪分析细胞因子 IL-10和 FoxP3的表达。 A， 淋巴结； B， 脾脏。
图 12b为共免疫诱导的调节性 T细胞表达 FoxP3。 第二次免疫后第 7天分离 T细胞 并用流式细胞仪分析细胞因子 IL-10和 FoxP3的表达。 A， 淋巴结； B， 脾脏。
图 13为共免疫没有改变 CD4+CD25+细胞的数量。 从免疫不同组合的小鼠中分离 T细 胞， 抗 CD4和抗 CD25的单克隆抗体进行染色， 用流式细胞仪进行分析。 A， 淋巴结； B， 脾脏。
图 14为质粒 T- M0G352C的酶切鉴定结果。
图 15为质粒 pVAXM0G352c的酶切鉴定结果。
图 16为质粒 pVAXM0G352c在 BHK21细胞的瞬时表达结果。
图 17为免疫诱导小鼠 EAE模型的发病情况， 纵坐标为发病指数， 横坐标为天数。 图 18为阴性小鼠经过继转移发病小鼠脾脏来源的 T细胞后， 在皮下一次免疫 CFA 佐剂乳化的 M0G抗原 200ug后的发病情况， 纵坐标为发病指数， 横坐标为天数。 图 19为阴性小鼠经过继转移发病小鼠的淋巴结来源 T细胞， 在皮下一次免疫 CFA 佐剂乳化的 M0G抗原 200ug后的发病情况， 纵坐标为发病指数， 横坐标为天数。
图 20为诱导小鼠发病后针对自身 M0G抗原的 T细胞扩增情况。
图 21为诱导小鼠发病后针对自身 M0G抗原的 T细胞内部表达一些细胞因子的情况。 实施发明的最佳方式
下述实施例中的实验方法， 如无特别说明， 均为常规方法。，
实施例 1、 预防和 /或治疗 I型糖尿病的疫苗
本实施例中预防和 /或治疗 I型糖尿病的疫苗有 4种： 1 ) 由人胰岛素蛋白 （Sigma 公司， 1-9278)和 pVAX- insulin组成， 2) 由人胰岛素中 B链上的 9一 23蛋白（B9- 23) 和 pcDB9-23组成， 3) 由人胰岛素蛋白 (Sigma公司， 1-9278) 和 pcDB9- 23组成， 4) 由 B9- 23和 pVAX- insulin组成。
B9-23的氨基酸序列是： S H L V E A L Y L V C G E R G (序列 1 ) 。 胰岛素中 B 链上的 9一 23蛋白（B9- 23)由北京 aoke公司合成。
其中， pVAX- insulin和 pcDB9-23按照如下方法构建：
取人胰腺组织， 利用购自 Takara公司的 RNA提取试剂盒， 在 TrizoL试剂中充分研 磨， 并根据说明书进行总 RNA的提取。 根据已发表的基因序列设计引物， 引物序列为: 扩增人胰岛素 cDNA基因的引物：
mlnsulinpl atggccctgttggtgcacttcctac
mlnsulinp2 ttagttgcagtagttctccagctgg
扩增人胰岛素 B链上自氨基末端第 9至 23位氨基酸残基组成的多肽 B9- 23的引物：
B9-23pl： agcaggaagcctatcttccaggtta
B9-23p2： gcagaggggtaggctgggtagtggt
B9-23的氨基酸序列是： S H L V E A L Y L V C G E R G 。 · ' 依照 TaKaRa RNA PGR Kit操作指南进行， 反转录用 Oligo (dT) 作下游引物， 反 应条件： 42°C30min，99°C5min, 5°C5min。 PCR反应用基因序列设计特异性引物， 扩增参 数： 94°C2min; 94°C30s, 55。C30s， 72°C70s 35个循环； 72°C 10min, 反应产物进行 1% 琼脂糖凝胶电泳检测，结果表明人胰岛素的 RT-PCR产物得到一条约 750bp的条带， B9 - 23 的 RT- PCR产物得到一条约 250bp的条带。
利用 Takara公司 PCR Fragment Recovery Kit 按其说明书进行 PCR产物的回收。 回收的 cDNA片段与 pMD 18- T载体（购自 Takara公司）在 T4 DNA连接酶的作用下 16 °C过夜， 使人胰岛素的 cDNA或 B9- 23的编码序列连接到 pMD 18-T的载体上。 连接产物 转化感受态细胞， 感受态细胞的制备及转化和质粒的提取按参考文献 (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, (2nd ed) . New York : Cold Spring Harbor Laboratory Press, 1989. 19-56)。 提取后的质粒用 EcoR I 和 Hind III双酶切进行重组质粒的鉴定， 酶切反应结束后进行琼脂糖凝胶电泳鉴定， 正确的克隆进行质粒的大量提取。 为鉴定克隆的 cDNA序列， 对重组质粒进行纯化， 并 用 BcaBEST primer RV-M和 BcaBEST primer Ml 3- 47对 cDNA在 PE377全自动测序仪进 行双向 DNA序列测定， 所得序列用 PE公司 SeqEd vl. 0. 3软件进行分析。 将经酶切和测 序鉴定含有核苷酸序列是 GenBank Accession Number AY899304的自 5'端第 56位至 388 位脱氧核糖核苷酸的人胰岛素 cDNA基因的质粒命名为 pMD- insulin,含有核苷酸序列是 GenBank Accession Number AY899304的自 5'端第 152位至 196位脱氧核糖核苷酸的 B9-23的 cDNA基因的质粒命名为 pMD- B9- 23。
用 Ec I和 Iho I从 pMD- insulin和 pMD-B9- 23分别切下人胰岛素基因片段和 B9-23 片段， 人胰岛素基因片段连接到同样酶切的真核表达载体 pVAXl (Ir itrogen公司）上， B9-23片段连接到同样酶切的真核表达载体 pcDNA3. 0 ( Ir itrogen公司）上，重组质粒 用 EcoR I和 Xho I进行双酶切鉴定和测序鉴定。 将经酶切和测序鉴定含有核苷酸序列 GenBank Accession Number AY899304的自 5'端第 56位至 388位脱氧核糖核苷酸的人 胰岛素脱氧核糖核苷酸的人胰岛素 cDNA基因的质粒命名为 pVAX-insulin, 含有核苷酸 序列 GenBank Accession Number AY899304的自 5'端第 152位至 196位脱氧核糖核苷酸 的 B9- 23的 cDNA基因的质粒命名为 pcDB9- 23。
pVAX-insulin的 EcoR I和 Xho I酶切鉴定结果如图 la所示， 0. 7%琼脂糖凝胶电泳 表明 pVAX- insulin经 EcoR I和 Xho I酶切后， 得到约 750bp的人胰岛素 cDNA基因片 段。 图 la中， 1 : DNA标准分子量（10000bp、 5000bp、 2500bp、 1000bp和 250bp， 购 自 Takara公司） ； 2, 3 : 重组质粒 pVAX- insulin用 EcoR I和 Xho I进行酶切。
pcDB9-23载体的 EcoR I和 Xho I酶切鉴定结果如图 lb所示， 0. 7%琼脂糖凝胶电泳 表明 pcDB9-23经 EcoR I和 Xho I酶切后， 得到约 250bp的 B9-23 cDNA基因片段。 图 lb中， 3是 DNA标准分子量（2000bp、 lOOObp, 750bp、 500bp、 250bp和 100bp，购自 Takara 公司） ； 2 :重组质粒 pCDB9- 23用 EcoR I和 Xho I进行酶切。
pVAX- insulin参照 lipofectaraine产品说明书利用脂质体转染法转染 BHK21细胞（美 国 ATCC公司） 。 转染细胞 48 h后收集细胞， 提取总 RNA以 mlnsulinpl和 mlnsulinp2为引 物用 RT-PCR方法检测目的基因的表达。 结果如图 lc所示， 表明转染细胞中有 750bp的人 胰岛素 cDNA基因片段， 说明 pVAX-insulin在 mRNA水平能有效在体外表达。 1 : DNA标准分 子量（10000bp、 5000bp、 2500bp、 lOOObp和 250bp，购自 Takara公司）； 2、 3： pVAX- insulin 转染的细胞 insulin的表达； 4: 未转染的细胞对照。
实施例 2、 预防和 /或治疗 I型糖尿病的疫苗的效果试验
分别免疫实验用的 Balb/c小鼠和 N0D小鼠。肌肉注射。 Balb/c小鼠每组 3只。 N0D 小鼠每组 16只。
一、 Balb/c小鼠实验
1、 在 Balb/c小鼠上的细胞免疫反应
Balb/c小鼠 9只， 均分为 3组， 每组 3只， 第 1组 （pcDB9- 23免疫组）每只分别 免疫含 100微克 pcDB9-23的 0. 9% NaCl 水溶液 100微升， 第 2组 (B9-23免疫组）每 只分别免疫含 100微克 B9- 23的 0. 9% NaCl水溶液 100微升，第 3组（pcDB9_23和 B9-23 共免疫组）每只分别免疫含 100微克 pcDB9- 23和 100微克 B9- 23的 0. 9% NaCl 水溶液 100微升。 第一次免疫后第 14天再以同等剂量加强免疫一次， 并于第二次免疫后第 7 天按如下方法进行 τ细胞增殖实验。
利用 T细胞增殖试验， CFSE染色，流式细胞仪检测，用来反映 T淋巴细胞针对特定 抗原的增殖能力。集体内淋巴细胞在收到抗原等特异或非特异的刺激后，导致细胞活化， 细胞因子合成、细胞因子受体表达及活化的细胞发生增殖。细胞增殖反应在一定的程度 上可以反映细胞的功能状态。
具体方法如下： 1， 脱臼处死小鼠， 用 70%乙醇浸泡 15分钟； 2， 在提前紫外灯灭菌 20分钟的超净工作台中， 无菌条件下取出小鼠脾脏于提前加有 2ml RPMI1640培养液的 细胞培养皿中； 3， 将铜网灼烧后降温放入平皿中， 利用无菌注射器将脾脏磨碎， 制成 细胞悬浮液， 并过滤到 13ml细胞离心管内； 4， 将离心管口用封口膜封好， 离心 2000 转， 10分钟； 5， 弃上层培养液， 加 2〜3ml红细胞裂解液， 悬浮细胞， 裂解 2分钟后， 加等体积 RPMI1640培养基（或胎牛血清） 中止反应， 将离心管口用封口膜封好， 离心 2000转， 10分钟； 6, 弃上层培养液， 加 3〜4ml RPMI1640 (含 2%胎牛血清）培养基悬 浮细胞； 7, 用玻璃棉 37°C慢慢滤过细胞， 保证细胞充分和玻璃棉结合以除去 B细胞； 8，用血球计数板细胞计数； 9，用 PBS洗掉培养基， 并最终用 PBS溶液 1ml悬浮 2X 107 个细胞； 10， 加入 3uM CFSE储备液至终浓度为 1. 5uM, 室温下轻轻振荡 8分钟； 11, 加 入等体积胎牛血清终止反应， 将细胞放入水浴 10分钟， 2000rpm 5分钟离心， 弃上清， 悬浮细胞， 并用 lml每 106个细胞的含有 PBS溶液洗细胞， 离心弃上清， 重复 3次； 12, 将每组细胞悬液分 4份加入 96孔培养板中。 其中一份加入 ΙΟΟμΙ Con A (有丝分裂原） 至终浓度为 5 g/ral， 一份加入相应的特异性抗原 （B9-23) 作为刺激物至终浓度为 5μ§/πι1, 一份不加刺激物， 一份加入 ΙΟΟμΙ BSA至终浓度为 2 g/ml作为无关抗原。 同 时设有不加刺激物和不用 CFSE染色的细胞对照； 13，将细胞放入细胞培养箱， 37°C, 5% C02 培养， 分别在 48， 96小时用流式细胞仪检测细胞增殖情况。
48小时和 96小时的检测结果表明， pcDB9-23和 B9-23共免疫组的 T细胞增殖程度 (增殖 0. 08% )明显低于 pcDB9- 23免疫组（增殖 22. 32% )和 B9- 23免疫组（增殖 8. 94 ) ， 证明 pcDB9- 23和 B9-23共免疫组出现了免疫抑制现象。 图 2a和图 2b中的百分 数为增殖程度， 数值越大表明增殖程度越高， Ml表示细胞增值的百分比。
2、 细胞免疫反应的特异性
为了进一步探索免疫抑制反应是否具有专一性， 即只有对胰岛素抗原的蛋白 和 DNA之间才会产生抑制现象。 将 Balb/c小鼠 21只， 均分为 7组， 每组 3只， 第 1 组（阴性对照组）每只分别免疫 0. 9% NaCl水溶液 100微升，第 2组（pVAX- insulin 免疫组）每只分别免疫含 100微克 pVAX-insulin的 0. 9% NaCl水溶液 100微升， 第
3组（人胰岛素蛋白免疫组）每只分别免疫含 100微克人胰岛素蛋白的 0. 9% NaCl水 溶液 100微升， 第 4组 （pVAXl免疫组）每只分别免疫含 100微克 pVAXl的 0. 9% NaCl 水溶液 100微升， 第 5组（pVAX- insulin和 VP1共免疫组）每只分别免疫含 100微克 pVAX-insulin和 100微克口蹄疫病毒 VP1 (按照文献： 金华利，张富春,单文娟，张 爱莲，李轶杰,王宾. 口蹄疫 vpl 蛋白在酵母中的表达及免疫原性分析. 细胞与 分子免疫学杂志， 2004年， 20 ( 5) 513- 516中描述的方法制备） 的 0. 9% NaCl水 溶液 100微升， 作为对照组； 第 6组（pVAXl和人胰岛素蛋白共免疫组）每只分别 免疫含 100微克 pVAXl和 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 7 组 （pVAX-insulin和人胰岛素蛋白共免疫组）每只分别免疫含 100微克
pVAX- insulin和 100微克人胰岛素蛋白的 0. 9% NaCl水溶液 100微升。 第一次免疫 后第 14天再以同等剂量加强免疫一次， 并于第二次免疫后第 7天按找上述步骤 1的 方法进行 T细胞增殖实验。 刺激后 48小时的结果如图 3a和 3b所示， 表明只有当胰 岛素蛋白与其对应的 DNA混合在一起时（pVAX-insulin和人胰岛素蛋白共免疫
组） ， 才出现免疫抑制现象， 其它混合组与对照组比较， T细胞活性没有明显的 下降。说明共免疫是同种抗原的蛋白与 DNA之间存在免疫抑制现象的特异性关系。
图 3a中的百分数为增殖程度，数值越大表明增殖程度越髙， Ml表示细胞增值的百 分比。 图 3b中， pi表示 pVAX-insulin免疫组， Insulin表示人胰岛素蛋白免疫组， pVAX表示 pVAXl免疫组， pI+VPl表示 pVAX- insulin和 VP1共免疫组， pVAX+In表示 pVAXl和人胰岛素蛋白共免疫组， pl + In表示 pVAX- insulin和人胰岛素蛋白共免 疫组， naive表示阴性对照组。
3、 细胞免疫反应的剂量关系实验
为了进一步探索抑制现象出现最佳效果时蛋白与 DNA之间的剂量关系。 将 Balb/c 小鼠 24只， 均分为 8组， 每组 3只， 第 1组（阴性对照组）每只分别免疫 0. 9% NaCl水 溶液 100微升，第 2组（pVAX- insulin免疫组）每只分别免疫含 100微克 pVAX- insulin 的 0. 9% NaCl 水溶液 100微升， 第 3组（人胰岛素蛋白免疫组）每只分别免疫含 100 微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 4组 （pVAX- insulin和人胰岛素 蛋白 1: 4共免疫组） 每只分别免疫含 25微克 pVAX-insulin和 100微克人胰岛素蛋白 的 0. 9% NaCl水溶液 100微升， 第 5组 (pVAX- insulin和人胰岛素蛋白 1： 2共免疫组） 每只分别免疫含 50微克 pVAX- insulin和 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 6组（pVAX- insulin和人胰岛素蛋白 1: 1共免疫组）每只分别免疫含 100 微克 pVAX- insulin和 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 7组
(pVAX-insulin和人胰岛素蛋白 2: 1共免疫组）每只分别免疫含 200微克 pVAX-insulin 和 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 8组 （pVAX-insulin和人 胰岛素蛋白 4: 1共免疫组）每只分别免疫含 400微克 pVAX- insulin和 100微克人胰岛 素蛋白的 0. 9% NaCl 水溶液 100微升。 第一次免疫后第 14天再以同等剂量加强免疫一 次， 并于第二次免疫后第 7天按找上述步骤 1的方法进行 T细胞增殖实验。 剌激后 48 小时的结果如图 3c所示， 当质粒与蛋白之间的比例是 2: 1, 抑制现象最明显。 图 3c 中， na'ive表示阴性对照组， pi表示 pVAX- insulin免疫组， In表示人胰岛素蛋白免疫 组， 1 :4、 1 :2、 1 : 1、 2 : 1、 4: 1分别表示 pVAX- insulin和人胰岛素蛋白 1: 4共免疫组、 pVAX-insulin和人胰岛素蛋白 1: 2共免疫组、 pVAX- insulin和人胰岛素蛋白 1 : 1共 免疫组、 pVAX-insulin和人胰岛素蛋白 2: 1共免疫组和 pVAX-insulin和人胰岛素蛋白 4： 1共免疫组。
二、 NOD小鼠上共免疫预防实验 将 64只雌性 NOD小鼠均分为 4组， 每组 16只， 第 1组（阴性对照组）每只分别免 疫 0. 9% NaCl 水溶液 100微升， 第 2组 （pVAX- insulin免疫组） 每只分别免疫含 100 微克 pVAX- insulin的 0. 9% NaCl 水溶液 100微升， 第 3组 （人胰岛素蛋白免疫组） 每 只分别免疫含 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升， 第 4组
(pVAX-insulin和人胰岛素蛋白共免疫组）每只分别免疫含 100微克 pVAX- insulin和 100微克人胰岛素蛋白的 0. 9% NaCl 水溶液 100微升。 第一次免疫后第 14天再以同等 剂量加强免疫一次， 并于第二次免疫后每周用血糖仪（购自北京怡成公司）检测小鼠的 血糖变化并记录。若连续两周小鼠的血糖水平超过 200mg/dl，即认为此 NOD患有糖尿病。
如图 4所示， 统计每组 NOD小鼠的发病率， 正常情况下， 雌性 NOD小鼠的糖尿病的 发病率是 60%左右。 在 pVAX- insulin和人胰岛素蛋白共免疫组里， NOD小鼠的发病率 有了明显的将低， 只有一只小鼠发病， 并且发病的时间也有了明显的延迟。 说明共免疫 pVAX- insulin和人胰岛素蛋白可有效地预防 I型糖尿病。 图 4中， Naive NOD表示阴性 对照组小鼠， Insulin表示人胰岛素蛋白免疫组小鼠， pVAX-insulin表示 pVAX- insulin 免疫组小鼠， Insulin+pVAX-insulin表示 pVAX- insulin和人胰岛素蛋白共免疫组小 鼠。
对阴性对照组小鼠、 已发病的糖尿病小鼠和 pVAX- insulin和人胰岛素蛋白共免疫 组的胰腺组织进行切片、 HE染色， 结果也表明， 在 pVAX- insulin和人胰岛素蛋白共免 疫组的胰岛组织中，淋巴细胞的浸润比其它发病组的情况要轻微，与正常的未发病的 N0D 小鼠 （阴性对照组小鼠）相近（图 5) 。
该实验证明了共免疫胰岛素 DNA和蛋白， 能够有效地预防 I型糖尿病的发生。 在本 次实验中重复验证了共免疫现象的免疫抑制性。 尤其在 N0D小鼠上， 能够预防 I型糖尿 病的发生。 ·
总之， 该实验证明了一种预防 I型糖尿病的新方法。 进一步探讨相关机制可能会发 展出对人和动物自身免疫疾病治疗的新方法。
实施例 3、 采用共免疫的方法治疗 /预防自主性卵巢炎
摘要 自身免疫卵巢疾病 （A0D: autoimmune ovarian disease) 是人类早期卵巢 败育（P0F: premature ovarian failure) 的诱因之一。 A0D的自身蛋白抗原有卵透明 带蛋白 （ZP3) 。
用 MZP3蛋白和 CFA诱导的 A0D小鼠模型， 我们探讨了一个更实用的治疗卵巢炎的技 术。根据我们以前观察到的结果一共免疫匹配的 DNA和蛋白可以诱导 T细胞反应的免疫抑 制， 我们采用共免疫的方法来改善 A0D。 结果显示， 共免疫 MZP3 DNA和蛋白改善了 A0D， 并且抑制了抗原特异性的 T细胞反应， 减低了炎症因子 IL- 12和 INF- y的表达水平， 这些 作用可能是由于共免疫诱导出了一群特异性的调节性 T细胞来完成的。这群调节性 T细胞 的特征是 CD47CD257FoxP3+， 并能表达 IL- 10。 我们第一次报道了用共免疫 DNA和蛋白的 方法来治疗自身免疫疾病。
近百年来， 近年来研究发现免疫系统和生殖系统之间存在着密切而复杂的关系 [1'2]。 据此关系建立了新型免疫不育技术， 该技术是利用基因重组等分子生物学技术为基础， 以生殖过程中的关键因子为靶抗原的疫苗，利用免疫系统对外加的靶抗原产生相应的免 疫应答， 使体内的靶生殖抗原丧失功能， 阻断过度繁殖动物的生育力， 破坏过度繁殖动 物的正常生殖过程， 维持生物物种之间及其与环境之间的动态平衡 [3]。 鼠的卵膜外都有 由一层卵透明带所完全包围。卵透明带是在初级卵母细胞成熟过程中的早期， 由卵母细 胞和初级卵母细胞分泌而形成的一层含糖蛋白的嗜酸性膜， 由 ZP1、 ZP2、 ZP3组成 [4]。卵 透明带在受精过程中有两个重要作用： 一是使精子附着在卵透明带上， 二是在精子结合 以后诱导精子的顶体反应使精子进入卵中 [s],其中 ZP3是 ZP中的一种主要糖蛋白，作为精 子的初级受体， 在精卵结合过程中更为重要， 已被认为是一种理想的不育疫苗 [6]。
但是在免疫不育疫苗的制备工程中发现常伴随卵巢炎的发生， Dunbar等 [7]用天然的 猪 ZP糖蛋白和去糖基的 ZP蛋白主动免疫雌性个体而导致阻断受精。但是， 研究表明免 疫后的动物在排卵周期、 激素水平和卵巢中卵泡发育方面， 有的出现了短暂的变化， 有 的出现了不可逆的改变。
妇女更年期的正常年龄一般为 50岁，如果在 40岁以前丧失了卵泡的功能就可被认 为是患有早期卵巢败育（POF: premature ovarian failure) , 这种疾病在妇女中的发病 率一般为 1-2%， 严重的可能在十几岁就会患这种疾病[8]。 自身卵巢免疫疾病 (A0D: autoimmune ovarian disease)被认为是 POF的主要诱因之一[9_11]； Rhim等采用脚底和皮 下注射 MZP3的一段短肽 (330 to 342)建立了一个新的小鼠实验动物模型用于卵巢自身 免疫疾病的研究， 这个模型可在一定程度上模拟妇女早期卵巢败育 [12]。这种自身免疫疾 病是由 CD4+ T细胞介导的， 并且 Lou等发现自身抗体的产生可以改变 T细胞介导的炎 症反应的分布， 并且会导致靶器官功能单位的丧失 [13]。
作为机体免疫系统的调控者——调节性 T细胞可以负调控机体对自身抗原的免疫反 应， 从而使机体免受自身免疫疾病的伤害。 天然的调节性 T细胞一般为 CD4+CD25+双阳 性 T细胞， 这类细胞可以表达一种功能性转录因子—— FoxP3, 这种细胞因子是正常免 疫系统重要的细胞组成成分【14]。 这类细胞的存在使得研究者使用抗原特异性的调节性 T 细胞去治疗自身免疫疾病和同种异体移植排斥 [ 17]。 Samy等使用抗原依赖性的
CD4+CD25+双阳性调节 T细胞控制了自身免疫疾病——卵巢炎的发生 [18〕。 Jin等发现采用 蛋白和 DNA共免疫的方法可以抑制抗原特异性的细胞反应 [19]，这种抑制功能可能与诱导 出了抗原特异性的调节性 T细胞有关， 所以本论文以小鼠为动物模型， 利用蛋白和 DNA 共免疫抑制细胞免疫反应的方法探索治疗自身免疫疾病——卵巢炎的方法及相应的机 理。
1 材料和方法
1. 1 材料和试剂
PMD18-T测序载体购自 Takara公司，大肠杆菌 DH5 菌株为我们实验室保藏菌 种。 RNA提取试剂盒、 PCR产物回收试剂盒、 DNA marker、 限制性内切酶、 exTaq酶以及 RT-PCR和 PCR引物均购自 Takara公司； 测序试剂盒购自美国 PE公司，其它试剂均为分 析纯。
1. 2 mzp3基因的克隆 取性成熟小鼠卵巢， 在 Trizo中充分研磨， 并根据说明书进行总 RNA的提取。 根据 已发表的基因序列 （GenBank number : BC103585) 设计引物， 引物序列为：
MZP3 PI : AATGAATTCATGAATTCCCAGACTCTGTGGC
MZP3 P2: TTACTCGAGTTAAGTCCAGCCTTCCACAGTCT
扩增片段为 MZP3的核酸序列第 63 到 1143位碱基。
依照 TaKaRa RNA PCR Kit操作指南进行， 反转录用 Oligo (dT) 作下游引物， 反 应条件： 42°C30min，99°C5min, 5°C5min。 PCR反应用 mzp3基因序列设计特异性引物， 扩 增参数： 94°C2min; 94°C30s, 55°C30s， 72°C70s 35个循环； 72°C 10min, 反应产物进 行琼脂糖凝胶电泳检测。 按 Takara公司 PCR Fragment Recovery Kit 说明书进行 PCR 产物的回收。 回收的 mzp3 cDNA片段与 PMD 18- T载体在 Τ4 DNA连接酶的作用下 16°C 过夜， 使 mzp3 cDNA连接到 pMD 18- T的载体上。 连接产物转化感受态细胞， 感受态的 制备及转化按参考文献 (Sambrook J等 1989) [2°]。 按相关的参考文献 (Sambrook J等 1989)进行质粒 DNA的小量提取， 提取后的质粒用 BamH I和 Hind III双酶切进行重组 质粒的鉴定， 酶切反应结束后进行琼脂糖凝胶电泳鉴定， 正确的克隆进行质粒的大量提 取。 为鉴定克隆的 cDNA序列， 对重组质粒进行纯化， 并用 BcaBEST primer RV-M和 BcaBEST primer M13- 47对 mzp3 cDNA在 PE377全自动测序仪进行双向 DNA序列测定， 所得序列用 PE公司 SeqEd vl. 0. 3软件进行分析。
1. 3真核表达载体的构建及组质粒在 BHK21细胞中的瞬时表达
将上述经 Ec I和 X w I双酶切， 连接到同样酶切的真核表达载体 pcDNA3， 该载体 命名为 pcDmzp3， 经序列鉴定正确的重组质粒进行真核转染实验。 具体转染方法参照 lipofectamine产品说明书。 利用脂质体转染法将 pcDmzp3转染 BHK21细胞， 具体操作步 骤见说明书。 转染细胞 48 h后收集细胞， 提取总 RNA以 MZP3 P1和 MZP3 P2为引物用 RT- PCR 方法检测目的基因的表达。
1. 4原核表达载体的构建、 蛋白表达及蛋白纯化
重组质粒 pMD18_T/ MZP3经 Eco/P I和 ho I双酶切， 连接到同样酶切的真核表达 载体 pGEX- 4T-1 , 经序列鉴定正确的重组质粒进行蛋白表达。 挑取单个菌落， 接种于新 鲜的 LB (含 AmP+ 50 mg/L)培养基中， 于 37 Ό培养过夜。 次日， 按 1%的接种量转种于新 鲜的 LB (含 Amp+ 50 mg/L) 培养基中。 当 A600达到 0. 6〜0. 8时， 在 1. 0 mraol/L IPTG 的诱导下，于 37°C表达 5 h。取 1 mL菌液离心收集菌体，再用双蒸水洗 1次，悬于 lxSDS 样品加样液中， 置沸水浴中煮 10 mine 以 12 000g离心 2 min, 取 15 μ L上清液进行 100 g/L SDS-PAGE, 并以考马斯亮蓝染色。
离心收集诱导后的 C0U BL21 (DE3) , 用 PBS洗涤后，用 1/20原培养体积的细胞 裂解液 Ll (10 mmol/L Tris-HCl (pH 8. 0)、 1 mmol/L EDTA和 200 mmol/L NaCl)重悬细菌 沉淀，冰浴 15 min后，超声裂解细菌至菌液不再粘稠。 然后 4°C， 12 000 g离心 10 min去上 清，收集沉淀获得初制的 MZP3包涵体蛋白。 将初制包涵体蛋白用 L1和 4M尿素洗涤， 70°C 保温 10min， 4°C, 12 000 g离心 5 rain, 重复 3次。 用 L3 ( 10 mmol/L Tris-HCl, 1 mol/L NaCl、 8M尿素、 5 mmol/L β -巯基乙醇和 5 mmol/L DTT, pHIO) 溶解包涵体蛋白， 55 °C 保温 10 min, 4°C， 12 000 g离心 5 min去除沉淀， 上清装入透析袋中在含 4M尿素的 L2 ( 10 mmol/L Tris-HCl (pH 8. 0)和1 mol/L NaCl ) 中透析 8h， 之后在含 2M尿素的 L2中透 析 8h，逐级递减尿素的浓度，直至将尿素从蛋白溶液中除去，用 Bradford测定蛋白含量。
1. 5卵巢炎模型的诱导
6-8周龄， 雌性， C57BL/6小鼠， 购于中国医学科学院实验动物研究所。 将浓度为
2mg/ml蛋白与完全傅式佐剂 （CFA: complete Freund' s adjuvant) 1： 1混合， 乳化完 全后每只小鼠通过脚掌和肌肉免疫 100 μ 1， 既每只小鼠免疫 100 蛋白。只免疫 CFA 的小鼠作为对照。 14天后采血， 收集血清， 间接 ELISA检测相应的抗体滴度。 以纯化的 GST-MZP3融合蛋白作为标准抗原， ELISA法检测小鼠血清中 ΜΖΡ3蛋白的特异性抗体水 平。将蛋白稀释成 5 μ g/ml , 96孔酶标板用 100 μ 1/孔包被 4 Ό过夜；弃去包被液 PBST 洗板 3次， 5%脱脂奶粉- PBST在 37Ό封闭 1 h; 洗板后加入不同稀释度的小鼠血清 37°C 孵育 2 h; 洗板后加入 1 : 1000辣根过氧化物酶标记的羊抗鼠二抗 （HRP-IgG) ， 50 μ 1/ 孔， 37 °C孵育 2 h后弃去； 洗板， 加入 50 μ L/孔 TMB室温避光显色反应 30min; 2mol/L 硫酸中止反应， 用酶标仪测定 OM50/650的值。 组织学评价卵巢疾病： 卵巢在 Bouin' s 固定液中固定 24小时， 进行石蜡包埋， 然后连续切片（5 μ m)并进行 H. E (hematoxylin and eosin)染色。 按严重程度对卵巢病理学进行分级， 1间质区域出现炎症； 2和 3增 加的多个位点的炎症反应或卵泡之间和内部的肉芽肿； 4卵泡消失和卵巢闭锁。 抗原特 异性淋巴细胞增殖反应： 免疫后 14d将小鼠处死， 无菌取出腿弯处淋巴节， 将淋巴节研 碎， 2000rpm离心 5min后弃去上清，用培养基重悬细胞； 用血球计数板计数后， 调整细 胞浓度到 lxl07mL。 取 2xl07个细胞， 2000rpm离心 5min后弃去上清， 用无菌 PBS重悬 细胞， 加入 1. 5 μ ΐ CFSE ( lmmol/ml ) ， 37°C轻摇 10min， 加入等体积的牛血清终止反 应， 2000rpm离心 5min后弃去上清， 用 PBS洗涤 3次； 最后用 1 ml培养基重悬细胞， 每空加 100 μ ΐ细胞， 用 ΜΖΡ3 ( 10 u g/ml )蛋白来刺激 T细胞增殖， 用 BSA (2 g/ml ) 作为非特异性抗原对照， ConA ( 10 g/ml ) 作为阳性对照， 用流式细胞仪检测增殖情 况。采用胞内染色的方法检测细胞因子 IL-12和 INF- y： 免疫后 14d将小鼠处死，无菌 取出脾脏， 将脾脏组织研碎， 2000rpm离心 5min后弃去上清，用 1- 2mL红细胞裂解液处 理细胞 2- 3min， 加 6-12mL含 4%血清的 RPMI 1640培养基终止， 2000rpm， 离心 5min后 弃去上清， 用培养基重悬细胞， 用血球计数板计数后， 调整细胞浓度到 2xl07mL。 淋巴 节单细胞悬液的制备同上， 每孔加入 2 χ10β个细胞（100 μ ΐ ) ， 用 10 g/ml的 ΜΖΡ3 蛋白刺激 4- 6个小时， 加入莫轮菌素（monencin)抑制 1-2小时。 然后进行胞内染色， 用流式细胞仪检测细胞因子的表达情况。具体过程如下： （1 )小鼠在免疫后 21天处死， 制备脾脏单细胞悬液， 加入 2 ml红细胞裂解液溶解红细胞， 用洗液洗一遍， 离心重悬 细胞进行计数； （2)抗 Fcg抗体封闭： 各组取 lxlO6个细胞， 加入适量抗 Fcg抗体（用 量见试剂说明书） ， 封闭 30分钟； （3)加入 2-3ml洗液， 2000rpm离心 5分钟， 弃去 上清， 各管加入适量荧光标记抗体充分混勾， 4°C避光反应 30分钟； （4) 加入 2- 3 ml 洗液， 2000rpra离心 5分钟， 弃去上清， 加入 0. 2 ml 4%多聚甲醛于 4°C固定 20分钟； (5)加入 2- 3 ml洗液， 2000rpm离心 5分钟， 弃去上清， 加入 0. 2 ml破膜剂细胞打孔， 充分混匀， 室温避光反应 15分钟； （6)加入 2- 3 ml洗液， 充分混勾， 2000rpm离心 5 分钟， 弃去上清， 各管加入适量的细胞内标记的荧光标记单克隆抗体（用量按试剂说明 书取用） ， 做细胞胞内分析物的标记； （7)细胞内染色抗体与细胞充分混匀， 室温避 光反应 30分钟； （8)加入 2-3 mL洗液， 充分混匀， 500xg离心 5分钟， 弃去上清， 每 管加入 300 洗液重悬细胞； （9) 流式细胞仪分析。 采用 RT-PCR方法检测细胞因子 IL-2:按照 TRIZOL Reagent ( Gibco )说明书,每组取 107个细胞，用 lml TRIzol Reagent 裂解， 然后加 200μ1氯仿， 混匀后， 12000r/min， 4°C离心 15min， 取上层水相转移至 新的 EP管， 加 500μ1异丙醇， 混勾后室温静置 10min， 12000r/min, 4°C离心 10min， 弃去上清， RNA沉淀用 75%的乙醇洗涤后， 溶于 20μ1经 DEPC处理的超纯水中， 一 80 °C储存。取 Ιμ ΝΑ样品加 599μ1超纯水，在紫外分光光度计上比色，读取 Α260和 Α280 的 0D值。根据试剂盒操作说明进行 RT- PCR，扩增后的产物用 1%的琼脂糖凝胶电泳检测。
1. 6共免疫治疗卵巢炎
将浓度为 2mg/ml蛋白与 CFA1 : 1混合， 乳化完全后每只小鼠通过脚掌和肌肉免疫
100 μ 1， 14 d后通过肌肉免疫 pcDmzp3+MZP3各 100 μ g， 免疫 pcDNA3、 pcDNA3+ MZP3、 pcDmzp3、 pcDmzp3+0VA和 MZP3作为对照组， 14 d后加强免疫， 在加强免疫后一周，无 菌取小鼠的脾脏细胞，通过 MTT法进行 T细胞扩增实验， MZP3蛋白抗原来剌激 T细胞增 殖， 用 BSA作为非特异性抗原对照， 用 ConA作为阳性对照。 具体过程如下： 用 MTT法 检测小鼠 T细胞体外增殖活性
( 1 )取脾脏： 加强免疫后 7d将小鼠处死， 无菌取出脾脏； （2)制成单细胞悬液: 将脾脏组织研碎， 2000rpm离心 5min后弃去上清， 用 1- 2mL红细胞裂解液处理细胞
2-3min， 加 6-12mL含 4%血清的 RPMI 1640培养基终止； 2000rpm， 离心 5min后弃去上 清， 用培养基重悬细胞。
(3)细胞计数： 用血球计数板计数后， 调整细胞浓度到 3xl07mL; (4)加入细胞 板： 每孔加入 ΙΟΟ μ Ι细胞， 分别加入 ConA (终浓度 10 g/ml ) ， MZP3蛋白 （终浓度 10 g/ml) , BSA (非特异性抗原， 终浓度 2 w g/ml)剌激 48- 72h; (5)显色： 每孔加 入 MTT溶液 20μί， 37。C， 5%C02,培养 3_4h， 2000 rpm离心 5min，弃上清，每孔加 100 μ 1 二甲基亚砜， 37°C轻摇 20-30 min; (6)读数：用酶标仪（Magellan, Tecan Austria GmbH) 测定 595nm的 0D值； （7) 结果计算： 刺激指数
SI= (各剌激孔的 0D值-培养基 0D值） I (未刺激孔的 0D值-培养基 0D值） 。 淋巴细胞扩增实验： 无菌条件下取小鼠腿弯处淋巴节， 将淋巴节研碎， 2000rpm离 心 5min后弃去上清， 用培养基重悬细胞制成单细胞悬液， 调整细胞浓度到 3xl07mL， 其余过程同上。
细胞因子 IL- 2、 IL - 12、 INFi、 IL- 10、 FoxP3和 CD25的检测及卵巢炎的评价同 上。
2 结果
2. 1 基因克隆及序列分析
采用提取的总 RNA ,以寡聚脱氧胸腺嘧啶为引物进行反转录得到单链 cDNA，再以设 计的 PCR引物进行扩增得到 MZP3基因片段。 RT- PCR产物经 1 %琼脂糖凝胶电泳鉴定， 可见一条约 1 200 bp的条带，与我们预计的一致。 RT- PCR产物与克隆载体 pMD18- T连 接，构建了重组质粒 pMD18- Τ/ MZP3。 重组质粒用 Bam A I和 Ηίηά III酶切，切出约 1200 bp DNA片段，与预计的连接片段大小相同。对 pMD18- T/ MZP3重组质粒用 BcaBESTprimer RV2M和 BcaBEST primer Ml 3247进行双向测序，测序结果显示与已发表的序列同源性为 99°/。。 用 DNAMAN对绒人、 猴、 狗、 猫、 猪、 和牛 MZP3基因进行同源性分析，其同源性 到达了 73% (图 6a) , 氨基酸序列同源性达到了 71% (图 6b)。
2. 2真核表达载体的构建及重组质粒在 BHK21细胞中的瞬时表达
重组质粒 PMD18- T/ MZP3及真核表达载体 pcDNA3用 EcoR I和 Xho I进行双酶切， 回收 纯化后进行连接， 将 MZP3基因克隆入真核表达载体 pcDNA3中获得重组载体 pcDmzp3。 重 组质粒以 EcoR I和 Xho I进行双酶切， 经 0. 7%琼脂糖凝胶电泳显示在大约 1200bp处有一 个条带 （图 7中 a) ， 说明重组质粒 pcDmzp3构建成功。 将纯化并定量好的质粒用脂质体 转染生长状态良好的 BHK21细胞，并且 72 h收集细胞， 以 MZP3pl和 MZP3p2为引物用 RT-PCR 方法检测目的基因的表达情况， 在 1200 bp处出现相应条带如图 7中13， 表明重组载体在 mRNA水平能有效在体外表达。
2. 3 原核表达载体的构建， 蛋白表达及纯化
重组质粒 PMD18- T/ MZP3及真核表达载体 pGEX4T- 1用 EcoR I和 Xho I进行双酶切， 回 收纯化后进行连接， 将 MZP3基因克隆入真核表达载体 PGEX4T- 1中获得重组载体 pGEX4T- 1/MZP3。 重组质粒以 EcoR I和 Xho I进行双酶切， 经 0. 7%琼脂糖凝胶电泳显示在 大约 1200bp处有一个条带 （见图 8中 a) ， 说明重组质粒 pGEX4T-l/MZP3构建成功。 将纯 化并定量好的质粒转化感受态细胞^: coii BL2KDE3) ,将构建的£ coii BL21 (DE3) /MZP3 转化子在新鲜 LB培养基中培养，经 IPTG诱导后收集转化菌。取其全菌蛋白进行 SDS-PAGE。 蛋白电泳结果表明， MZP3基因在£ ^ ' 8121 (0£3)中得到了大量表达（图 8中 b ) 。 收集 诱导表达后的菌体按超声裂菌处理。 SDS-PAGE结果表明诱导表达的蛋白质条带存在于细 菌裂解物的沉淀中， 说明蛋白是以包含体的方式表达的。我们用纯化包含体的方法对蛋 白进行了溶解， 洗涤和复性， 得到了纯度较高的蛋白 （图 8中 c ) 。
2. 4 自主免疫疾病——卵巢炎的诱导
将上述纯化的 MZP3蛋白的浓度调整到 2 mg/ml和等体积的完全弗氏佐剂 （CFA)完 全乳化， 通过脚掌和肌肉注射的方法免疫 8- 10周龄雌性 C57BL/6小鼠， 每只小鼠 100 μ 1， 注射 lOOul弗氏完全佐剂 (CFA)的小鼠为佐剂对照组， 注射 lOOul PBS为阴性对照 组。 14天后处死小鼠， 取出卵巢， 在 Bouin' s固定液中固定 24小时， 然后用石蜡包埋， 进行连续切片和 H. E ( hematoxylin and eosin)染色。 结果表明， 免疫 MZP3蛋白和 CFA 的小鼠 8只中有 7只出现了不同程度的卵巢炎， 发病率为 87. 5% (图 9a) 。 在卵巢的间 质和正在生长的以及成熟的卵泡中均发现了炎症反应。卵泡被炎症细胞浸润并且严重的 导致了卵子的丢失（图 9b ) 。 相比而言， 只注射 CFA的小鼠没有出现卵巢炎。
我们用 ELISA法对釆集的血清检测了抗 MZP3的抗体， 免疫 14天后， 与对照组相比 血清中出现了 MZP3抗体， 抗体滴度达到了 25600 (图 9c ) 。 免疫 MZP3蛋白和 CFA引起 了强烈的 T细胞反应（图 9d)， 细胞因子 IL- 2 (图 10a) ， INF- γ (图 10b) 和 IL-12 (图 10c) 的表达水平得到了提高， 这表明 Thl型的免疫反应被激活了。 这些结果显示 卵巢炎被成功诱导， 并且能够用来评价这种方法的治疗效果。
2. 5共免疫 MZP3 DNA和蛋白抑制了 A0D的发生
我们用构建的 A0D模型检测共免疫策略治疗卵巢炎的能力。 C57BL/6小鼠先通过脚掌 和肌肉注射 MZP3蛋白和 CFA, 14天后， 肌肉免疫 MZP3蛋白和质粒 pcDmzp3， 两周后加强免 疫。二免后 7天检测卵巢炎的发病情况。 结果发现， 只有共免疫质粒 pcDmzp3和 MZP3蛋白 (命名为 pcDmZp3+MZP3) 的小鼠才出现了 A0D的抑制现象（图 11a) 。 为了排除非特异 性因素的影响， 如质粒的骨架序列或无关蛋白等， 我们同时设立了免疫空质粒 pcDNA3和 MZP3蛋白共免 （命名为 pcDNA3+MZP3) 、 质粒 pcDmzp3和 OVA蛋白共免 （命名为 pcDmzP3+0VA)组作为对照。 结果显示对照组并没有出现 A0D的抑制现象， 说明只有共免 pCDmzp3+MZP3的小鼠才能抑制 A0D的发生， 进而说明这种抑制是抗原特异性的。 组织学 分析也揭示了免疫 PCDNA3+MZP3或 pcDmzp3+0VA的小鼠卵巢出现了炎症细胞浸润，而免疫 pcDmzp3+MZP3的小鼠卵巢没有炎症细胞浸润 （图 lib) 。
IL- 2、 IL- 12和 INF- γ髙水平表达是 Thl型 CD4+ T细胞反应的特点并且这些免疫调节 因子在几种自主免疫疾病中起了重要作用 [21— 26]。我们检测了共免 pcDmzP3+MZP3的小鼠这 些细胞因子的表达是否发生了变化。 免疫 pcDNA3+MZP3或 pcDmzP3+0VA的小鼠 IL- 12和 INF - γ的表达没有被抑制， 而免疫 pcDmzp3+MZP3 的小鼠 IL- 12 (图 11c)和 INF- γ (图 lid) 的表达被抑制。 这就暗示了共免 pcDmzp3+MZP3的小鼠出现了抗炎症免疫调节的功 能。
T细胞反应被认为参与了 A0D的发生， 所以我们检测了从小鼠脾脏中分离的 T细胞， 这些小鼠首先被免疫了 MZP3蛋白和 CFA， 14天后免疫 pcDNA3、 pcDNA3+MZP3、 pcDmzp3、 pcDmzp3+MZP3、 pcDmzp3+0VA和 MZP3, 两周后加强免疫， 在二免后第 7天取出脾脏。 这 些 T细胞被用来分析对 MZP3 蛋白抗原的反应能力。 从免疫 pcDmzp3+MZP3的小鼠中分离 的 T细胞基本上没有扩增能力， 而从其它组分离的 T细胞显示出了很强的扩增能力 （图 He) 。 这些结果显示共免疫 pcDmZp3+MZP3出现的 A0D抑制现象与没有抗原特异性的 T细 胞反应是相关的。 进一步说明共免疫 pcDmzp3+MZP3出现的 A0D抑制现象是通过下调炎症 因子的表达和抑制 T细胞反应产生的。 总的来说， A0D的抑制是抗原特异性的， 因为错 配的组合没有产生同共免疫 pcDmzp3+MZP3—样的结果。
MZP3自主抗体在指导自身免疫炎症反应的分布方面起到了重要作用并且能够增强 自身免疫疾病的严重程度 [13]。所以我们接下来检测了共免疫是否抑制了抗 MZP3抗体的产 生。 然而结果显示自主抗体的滴度在免疫组 pcDmzp3+MZP3、 pcDNA3+MZP3或 MZP3之间是 相同的 （图 l lf) 。 这个结果同以前的研究是一致的， 如果没有 T细胞反应只转移抗体 不能诱导自身免疫卵巢炎疾病 [13]。
2. 6 定性共免疫诱导的调节性 T细胞的特征
调节性 T细胞能够抑制 T细胞反应并且阻止自身免疫免疫疾病的发生。 细胞因子 IL-10和 FoxP3在 T细胞反应抑制过程中起到了重要作用[27— 29]。 为了检测由共免疫诱导 的调节性 T细胞能否表达特征性的细胞因子和标志， 我们从分别免疫了 pcDNA3、 pcDNA3+MZP3、 pcDmzp3 pcDmzp3+MZP3、 pcDmzp3+0VA和 MZP3的小鼠中分离出 T细胞。 这些细胞因子或标志用特异性的荧光标记的抗体进行胞内染色，通过流式细胞仪进行分 析。 从免疫 pcDmzp3+MZP3的小鼠中分离的 Τ细胞能够高水平表达 IL- 10 (图 12a)和 FoxP3 (图 12b ) ， 相反， 对照组只能表达低水平的 IL- 10和 FoxP3。 转录因子 FoxP3是 调节性 T细胞的特征性因子 [3°'31]，所以我们推测共免疫可能诱导出了一种调节性 T细胞。 我们检测了这类调节性 T细胞是不是属于 CD4+CD25+双阳性的， 对 CD25的表达情况进行 了检测， 发现各组之间 CD25的表达是基本相同的 （图 13 ) 。
综合以上数据， 采用共免疫相同基因的 DNA和蛋白我们可能诱导出了一类
CD4+CD25-调节性 T细胞， 这类 T细胞可以抑制抗原特异性的 T细胞反应并能阻止自身 免疫疾病的发生。
讨论
我们的研究证明了共免疫 MPZ3 DNA和蛋白诱导了一种调节性 T细胞，这种调节性 T 细胞可以抑制自身免疫疾病卵巢炎的发生。这种调节性 T细胞的表型为 CD4+CD25— Foxp3+， 表达 IL-10并且能够抑制抗原特异性 T细胞反应， 减少细胞因子 IL- 12和 INF- y 的表 达。
卵透明带蛋白在哺乳动物中是非常保守的，在小鼠中建立 A0D模型可以更好的理解 其它动物尤其是人类自身免疫疾病卵巢炎的发病机理。 通过脚掌和皮下注射 15个氨基 酸的 ZPP3短肽 (328-342) 与 CFA, 在 B6AF1 小鼠 [ (C57BL/6 X A/J) F1]上诱导出了 自身免疫疾病卵巢炎 [12' 13]， 而 C57BL/6小鼠没有卵巢炎的发生。通过在脚掌和肌肉注射 MZP3蛋白和 CFA， 我们在 C57BL/6小鼠上成功建立了卵巢炎模型， 尽管疾病的严重程度 不如以前报道的[13]。 推测以下两个原因可能造成了这一差异。 （1 )我们使用的 MZP3蛋 白含有更多的 T细胞表位， 这些细胞表位引起了更强的 T细胞免疫反应， 而 A0D就是由 T细胞反应引起的[12]。 （2)将皮下注射改为肌肉注射和更多的 B细胞表位诱导产生了更 高水平的自身免疫抗体。 自身免疫抗体可以改变 T细胞介导的炎症反应的分布并能加重 炎症反应[13]。
天然调节性 T细胞 CD4+CD25+可以抑制 T细胞反应并维持了自身免疫耐受。 如果将 调节性 T细胞去除则可引起疾病的发生[32' 33' u 所以这些细胞在阻止自身免疫疾病发生 中起到了重要的作用。这些特征使得调节性 T细胞成为了潜在的自身免疫疾病治疗工具 [35]。 最近是一些研究结果表明， 调节性 T细胞确实可以治疗自身免疫疾病， 这些调节性 T细胞可以是抗原非特异性的 [36— 38]， 也可以是抗原特异性的[18' 39]。 之前， 我们也报道过 共免疫 pcD- VP1 和 146S抗原诱导了 T细胞反应免疫抑制 [19]。 在这个研究中我们用这种 方法去抑制 A0D， 既共免疫 MZP3 DNA和蛋白。 抗原特异性的 T细胞反应被抑制， 并且细 胞因子 IL-12和 INF- γ的表达也被下调， 而 IL-10和 FoxP3的表达水平是上升的， 我 们推测可能诱导出了一类抗原特异性的调节性 T细胞。 CD25的染色结果表明，诱导的这 一类调节性 T细胞可能是 CD25-的。 因此我们得出结论， 共免疫诱导出了抗原特异性的 免疫抑制， 这种免疫抑制是由 CD4+CD25- FoxP3+IL10+调节性 T细胞介导的。 总之， 我们证明了一种改善自身免疫疾病的新方法。一类新的调节性 τ细胞可能被 诱导并介导了抗原特异性的 τ细胞免疫抑制。 因此， 进一步探讨相关机制可能会发展出 对人和动物自身免疫疾病治疗的新方法。
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38. Mottet C, Uhlig HH, Powrie F. Cutting edge : cure of colitis by CD4+CD25+ regulatory T cells. J Immunol. 2003 ; 170 : 3939-3943.
39. Tang Q, Henriksen KJ, Bi M, et al. In vitro- expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 2004 ; 199 : 1455-1465. 实施例 4、 采用共免疫的方法治疗 /预防多发型硬化症
多发性硬化（multiple sclerosis, MS)是中枢神经系统炎症性脱髓鞘疾病的代表， 属于自身免疫疾病。 研究 MS的最常见的动物模型称为 "实验性变应性脑脊髓炎"
(experimental allergic encephalomyelitis, EAE) ， 动物的 EAE临床表现及病理改 变与急性多发性硬化极其相似， 因此成功建立 EAE动物模型是研究 EAE的先决条件。
本实验题目的目的就是基于联合免疫引起的免疫抑制，在小鼠的 EAE模型中进行治 疗或预防 EAE的发生， 并在研究过程中进一步探讨 MS发病的相关免疫机制。
多发性硬化 （multiple sclerosis, MS) 的自身蛋白抗原有髓磷脂寡突细胞糖蛋白 (M0G)和二种髓磷脂抗原 （MBP和 PLP蛋白脂蛋白）。
目前的实验结果， 仅停留在 EAE小鼠模型的诱导及相关 M0G基因 DNA的构建上， 还 未深入展开， 初步结果总结如下。
1、 M0G35-55 2copies基因的克隆与表达
1. 1 材料和试剂
PMD18-T测序载体购自 Takara公司， 大肠杆菌 DH5 α菌株为我们实验室保藏菌种。 PCR产物回收试剂盒、 DNA marker、 限制性内切酶、 exTaq酶以及 RT- PCR和 PCR引物均 购自 Takara公司 ·' 测序试剂盒购自美国 PE公司，其它试剂均为分析纯。
1. 2方法
1. 2. 1 M0G35- 55基因克隆
诱导动物的 EAE模型常用的自身抗原为 M0G (Myelin Oligodendrocyte
Glycoprotein) 的 35- 55个氨基酸 （氨基酸序列为： MEVGWYRSPFSRVVHLYRNGK) ， 其 DNA 序列为： GAGGTGGGTTGGTACCGTTCTCCCTTCTCAAGAGTGGTTCACCTCTACCGAAATGGCAAG
根据基因序列， 设计合成引物及 DNA片断， 通过 Overlap PCR的方法构建两拷贝 M0G35-55的质粒 DNA。
Overlap DNA片断设计：
5' -ATGGAGGTGGGTTGGTACCGTTCTCCCTTC TCAAGAGTGG-3 '
5， -CCTCCATCTTGCCATTTCGGTAGAGGTGAACCACTCTTGAGAAGGGAGAA-3，
5 ' -CCGAAATGGCAAGATGGAGGTGGGTTGGTACCGTTCTCCCTTCTCAAGAG-3，
5， - TTACTTGCCA TTTCGGTAGA GGTGAACCAC TCTTGAGAAG GGAGAACGG-3'
5， - AAGGATCCATGGAGGTGGGTTG -3，
5，一 GCTCTAGATTACTTGCCATTTC— 3， 35s 35个循环； 72°C 10min, 反应产物进行琼脂糖凝胶电泳检测。 按 Takara公司 PCR Fragment Recovery Kit 说明书进行 PCR产物的回收。 回收的 M0G35- 55 2copies片段 与 pMD 18-T载体在 T4 DNA连接酶的作用下 16°C过夜， 使其连接到 pMD 18- T的载体上。 构建的质粒命名为： T-M0G352C连接产物转化 DH5 ct菌株的感受态细胞。小量提取质粒 并用 EcoRI I和 Hind III双酶切进行重组质粒的鉴定， 酶切反应结束后进行琼脂糖凝 胶电泳鉴定。 为鉴定克隆的 DNA序列， 对重组质粒进行纯化并进一步序列分析， 后应用 DNAMAN软件进行分析结果。
1. 2. 1真核表达载体的构建及组质粒在 BHK21细胞中的瞬时表达
将序列分析正确的质粒经 BamH I和 Hind III双酶 ί刀， 连接到同样酶切的真核 表达载体 pVAXl上， 该载体命名为 pVAXM0G352c， 经序列鉴定正确的重组质粒进行真核 转染实验。 具体转染方法参照 lipofectamine产品说明书。 利用脂质体转染法将 pVAXM0G352c转染 BHK21细胞。 转染细胞 48 h后收集细胞， 提取总 RNA以 M0G35 P1和 M0G35 P2为引物用 RT- PCR方法检测目的基因的表达。
1. 3 实验结果
质粒 T-M0G352c的酶切鉴定结果如图 14所示。 质粒 pVAXMOG352c的酶切鉴定结果 如图 15所示， 质粒 pVAXM0G352c在 BHK21细胞的瞬时表达结果如图 16所示。
2、 EAE动物模型的诱导及鉴定
2. 1直接免疫的方法：
C57BL/6小鼠， 背部皮下免疫用弗氏完全佐剂充分乳化的短肽 M0G35- 55
200ug (lug/ul，200ul，并且内含结合杆菌 BCG750ug)， 同时在免疫的第 0、 2天尾静脉 注射百日咳杆菌（ 108— 109个）， 7天后加强一次免疫 M0G200ug。
评分标准：
0——无任何临床症状。
1——动物尾部无力， 瘫软。
2——动物尾部无力， 前肢或后肢中等无力。
3——前肢或后肢严重无力， 人为翻身后不能恢复。
4——肢体麻痹， 人为翻身后不能恢复。
5——濒死状态。
结果如图 17 (不同颜色表示不同的小鼠个体） ： 40天后的较严重发病率 （发病指数 >2)可达 70%。
最近免疫的一批（20只） ， 初免后五周发病到 4的有 5只。
2. 2过继转移发病小鼠 T细胞诱导模型- 图 18为阴性小鼠经过继转移发病小鼠脾脏来源的 T细胞后， 在皮下一次免疫 CFA佐 剂乳化的 M0G抗原 200ug;
图 19为阴性小鼠经过继转移发病小鼠的淋巴结来源 T细胞， 在皮下一次免疫 CFA佐 剂乳化的 M0G抗原 200ug;
结论：过继转移淋巴结来源的 T细胞比脾脏来源的 T细胞小鼠发病早，症状变化稳定。 但转移脾脏 T细胞的小鼠发病指数较高。
2. 3发病小鼠的一些生理指标检测
图 20显示诱导小鼠发病后针对自身 M0G抗原的 Τ细胞扩增情况。
结论： 患病小鼠外周免疫器官有针对自身抗原 M0G的强烈的 Τ细胞扩增反应。 图 21显示诱导小鼠发病后针对自身 M0G抗原的 Τ细胞内部表达一些细胞因子的情 况。
3、 联合免疫治疗 ΕΑΕ小鼠 3. 1 策略
按照 2. 1的方法， 用 C57小鼠诱导 ΕΑΕ模型， 记录临床症状， 至所有小鼠的临床指数 达到 3以上水平。 将患病小鼠分组如下， 分别进行免疫， 免疫方法为肌肉注射。
1、 M0G35- 55肽 lOOul (与弗氏完全佐剂 1 : 1混合完全乳化至浓度 lug/ul )
2、 pVAXM0G352c质粒 DNA lOOul (浓度为 lug/ul )
3、 pVAXM0G352cM0G35- 55肽 +pVAXM0G352c 分别 lOOul (浓度同上）
4、 pVAXM0G352cM0G35- 55肽 +pVAXl分别 lOOul (浓度同上）
5、 不治疗组
14天后加强一次免疫。
自第一次治疗免疫起观察记录临床发病症状， 并评分。 加强免疫后 7天， 无菌取各实 验组小鼠脾脏， 通过 MTT法进行 T细胞扩增实验。 同时取小鼠大脑， 小脑， 脊髓用 4% 多聚甲醛固定组织， 进行组织切片的制作， 镜检病变情况。
IL- 1、 IL-2. INFi、 IL- 4、 IL- 10、 FoxP3、 IL17、 TGF 等细胞因子的检测： 分为 脾脏细胞细胞因子的表达情况以及脑脊液中细胞因子的表达情况两种。
下一步实验是在动物模型建立健全的基础上进行免疫学实验验证共免疫的应用。

Claims

权利要求
1、 一种预防和 /或治疗自身免疫疾病的疫苗， 它的活性成分是下述混合物： 由造成 5 自身免疫疾病的蛋白抗原或其表位多肽和在多克隆位点插入自身蛋白抗原或其表位多 肽编码基因的重组真核细胞表达载体组成的混合物； 所述自身蛋白抗原为胰岛素、谷氨 酸脱羧酶、 热休克蛋白、 髓磷脂寡突细胞糖蛋白、 二种髓磷脂抗原、 卵透明带蛋白 3、 肌球蛋白、 II型胶原蛋白、 甲状腺球蛋白、细胞膜表面抗原、第二型胶质抗原、 乙酰胆 碱受体、 甲状腺细胞表面抗原、 唾液腺管蛋白、 甲状腺球蛋白、 超抗原或感光器间受体 10 树脂样结合蛋白。
2、 根据权利要求 1所述的疫苗， 其特征在于： 所述预防和 /或治疗自身免疫疾病的 疫苗为预防和 /或治疗 I型糖尿病的疫苗；所述预防和 /或治疗 I型糖尿病的疫苗的活性 成分是下述混合物：
1) 由 I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原 15 编码基因的重组真核细胞表达载体组成的混合物；
2) 由 I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身 蛋白抗原表位多肽编码基因的重组真核细胞表达载体组成的混合物；
3) 由 I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗原 表位多肽编码基因的重组真核细胞表达载体组成的混合物；
20 4) 由 I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自身 蛋白抗原编码基因的重组真核细胞表达载体组成的混合物；
所述 I型糖尿病自身蛋白抗原为胰岛素或谷氨酸脱羧酶或热休克蛋白。
3、 根据权利要求 2所述的疫苗， 其特征在于： 所述胰岛素来源于人、 .狗或猫； 所 述谷氨酸脱羧酶来源于人、 狗或猫； 所述热休克蛋白来源亍人、 狗或猫。
25 4、 根据权利要求 3所述的疫苗， 其特征在于： 所述 I型糖尿病自身蛋白抗原为人 胰岛素。
5、 根据权利要求 3所述的疫苗， 其特征在于： 所述 I型糖尿病自身蛋白抗原表位 多肽的氨基酸序列是序列表中的序列 1。
6、 根据权利要求 2所述的疫苗， 其特征在于： 用于插入所述 I型糖尿病自身蛋白 -30 抗原编码基因或所述 I型糖尿病自身蛋白抗原表位多肽编码基因的真核细胞表达载体 为哺乳动物细胞表达载体。
7、 根据权利要求 6所述的疫苗， 其特征在于： 所述哺乳动物细胞表达载体为 pcDNA3. 0或 pVAXl或 provax。
8、 根据权利要求 7所述的疫苗， 其特征在于： 所述预防和 /或治疗 I型糖尿病的疫 35 苗的活性成分是人胰岛素蛋白和 PVAX- insulin。
9、 根据权利要求 7所述的疫苗， 其特征在于： 所述预防和 /或治疗 I型糖尿病的疫 苗的活性成分是 B9- 23和 pcDB9- 23。
10、 根据权利要求 2所述的疫苗， 其特征在于： 1)所述 I型糖尿病自身蛋白抗原 和在多克隆位点插入所述 I型糖尿病自身蛋白抗原编码基因的重组真核细胞表达载体 的质量比为 1:5- 5:1; 优选为 1:1- 1: 2；
2)所述 I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自 身蛋白抗原表位多肽编码基因的重组真核细胞表达载体的质量比为 1:5-5:1; 优选为 1:1-1： 2；
3)所述 I型糖尿病自身蛋白抗原和在多克隆位点插入所述 I型糖尿病自身蛋白抗 原表位多肽编码基因的重组真核细胞表达载体的质量比为 1:5-5:1; 优选为 1:1-1: 2；
4)所述 I型糖尿病自身蛋白抗原表位多肽和在多克隆位点插入所述 I型糖尿病自 身蛋白抗原编码基因的重组真核细胞表达载体的质量比为 1:5-5:1; 优选为 1:1- 1: 2。
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Publication number Priority date Publication date Assignee Title
JP2010259378A (ja) * 2009-05-08 2010-11-18 Hitachi Plant Technologies Ltd 細胞培養方法、細胞培養システム

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