WO2018107984A1 - 一种作为b细胞表位或肽半抗原的免疫刺激物的多肽和其构成的多肽免疫原，以及它们的用途 - Google Patents

一种作为b细胞表位或肽半抗原的免疫刺激物的多肽和其构成的多肽免疫原，以及它们的用途 Download PDF

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WO2018107984A1
WO2018107984A1 PCT/CN2017/114349 CN2017114349W WO2018107984A1 WO 2018107984 A1 WO2018107984 A1 WO 2018107984A1 CN 2017114349 W CN2017114349 W CN 2017114349W WO 2018107984 A1 WO2018107984 A1 WO 2018107984A1
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polypeptide
peptide
seq
amino acid
immunogen
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French (fr)
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李泰明
顾小骞
李志鑫
方金芝
魏晓敏
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a polypeptide comprising a T helper cell epitope (Th epitope) and use thereof, in particular, the polypeptide is used for a target antigenic site of a B cell epitope or a peptide hapten, a target antigen protein.
  • Th epitope T helper cell epitope
  • a polypeptide immunogen which acts as an immunostimulant for a B cell epitope or peptide hapten, enhances the immunogenicity of a target antigenic site (B cell epitope or peptide hapten) and can be used to prepare specific and a B cell epitope or an antibody to which a target antigen protein of a peptide immunogen binds, a polypeptide immunogen comprising the stimulating polypeptide, a coupled protein and a fusion protein, and a specific antibody produced based thereon, and a derivative and the like containing the same,
  • the drugs and preparations of specific antibodies and antibody derivatives and analogs produced on the basis of the invention have important application value in the prevention and treatment of diseases which are dominant in Th1, and specific antibodies, derivatives and analogs thereof can detect related target antigen proteins.
  • the present invention relates to the fields of immunology, pharmacy and medicine.
  • Th1 cells mainly produce Th1 cytokines IL-2, TNF- ⁇ and IFN- ⁇ ; Th2 cells mainly produce IL-4, IL-5 and IL-6. , IL-10 and IL-13.
  • Th1/Th2 balance drift It is customary to refer to the state in which Th1 and its cytokines predominate as the Th1 state, and the state in which Th2 and its cytokines predominate is referred to as the Th2 state.
  • Th1/Th2 affects the balance of cytokine network, which is closely related to the occurrence, development, treatment and outcome of many diseases.
  • Th1/Th2 balance disorder is different.
  • Rheumatoid arthritis type 1 diabetes, multiple sclerosis and chronic nail In diseases such as glandular gland, cytokines secreted by Th1 or Th1 predominate, Th1 induces onset and aggravates the condition; Th2 can alleviate the symptoms and even prevent the onset, which is related to the inflammatory and anti-inflammatory responses of Th1/Th2.
  • Th1/Th2 drift states and various diseases more and more studies are tending to discover and develop drugs and methods that can reverse or stabilize the Th1/Th2 state.
  • Epitopes are the basis of protein antigenicity. Protein antigens express their immune properties through epitopes. For a certain protein, it contains various epitopes such as B cell epitopes, also known as antigenic determinants (AD), T. Helper cell (Th) epitopes, etc., as well as inhibitory epitopes, toxic epitopes, cross-reactive epitopes, and the like. Selecting the appropriate epitope and linking it to other peptides or proteins, etc. by appropriate methods, and what adjuvants and dosage forms, how to immunize, etc., can obtain the desired polypeptide and protein vaccine, producing specific high titer (high titer) antibodies. It is hugely challenging.
  • Th1 produces IgG2a subtype antibody in mouse
  • Th2 type produces IgG1 and IgG2b subtype antibodies
  • Th1 produces IgG1 subtype antibody in human
  • Th2 type produces IgG2a subtype antibody
  • Th2 subtype antibody can increase Th2
  • the production of cytokines can treat high Th1 diseases and alleviate the symptoms.
  • the method of judging the Th1 or Th2 cell effect in the pathogenesis of disease immune disease is mainly based on the expression of local cytokines in inflammatory tissues, and can also be judged according to the changes of antibody subtypes in peripheral serum.
  • human infections and autoimmune diseases associated with high Th1 levels based on changes in Th1 or Th2 cell effects following various Th1 or Th2 cell effects have type 1 diabetes and complications. , kidney disease, nephritis, rheumatoid arthritis, reactive arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, nodular erythema, habitual abortion, etc.
  • B cell epitopes or peptide haptens The determination of B cell epitopes or peptide haptens is of great significance for the research of peptide vaccine synthesis, preparation of diagnostic reagents, screening of monoclonal antibodies, etc., but B cell epitopes or peptide haptens promote the body to produce high titer antibodies.
  • the premise is to have strong immunogenicity, usually in order to increase B cell epitopes or peptides
  • the immunogenicity of a hapten by coupling a B cell epitope or a peptide hapten to a protein such as KLH or BSA or a fusion protein of a macromolecule to increase the Th response, the method of constructing the fusion protein is time consuming, costly, and has a preparation cycle.
  • the antibodies produced by KLH and BSA immunization are more than Th1 antibodies, and the immunogens used to connect B cell epitopes or peptide haptens are not suitable for the treatment of high Th1 diseases such as type 1 diabetes and complications.
  • Th T helper
  • P277 peptide is a specific polypeptide of 437-460 of HSP60, which is a total of 24 amino acids. It is also an antigenic determinant that can react with effector T cells and is a specific fragment that plays a role in T1DM. DiaPep277 is currently entering Phase III clinical studies.
  • P277 peptide according to the same subcutaneous administration method of foreign DiaPep277 can cause damage of vascular endothelial cells and induce the production of antibodies to induce severe atherosclerosis; P277 is divided into P1 peptides ( The 437-450) and P2 peptides (448-460) have been shown to have a certain pro-atherogenic effect, while the P2 polypeptide is a Th2 epitope peptide in which P277 plays a major role in T1DM.
  • Islet cell-specific antigen-insulinoma associated protein-2 (IA-2) is considered to be one of the main target antigens for the initiation of autoimmune diabetes, type 1 diabetes mellitus (T1DM), mainly in the brain. Expression in the pancreas, pancreatic islet, and neuroendocrine cells.
  • the linear sequence of positions 626-630 of the proximal membrane region JM2 of IA-2 is a B cell epitope (626FEYQD630), which is named IA2 (5).
  • Immunization of model animals with IA-2 or B cell epitopes therein is a strategy for the prevention and treatment of type 1 diabetes.
  • one of the objects of the present invention is to provide a stimulating polypeptide comprising a T helper epitope (Th epitope) as an immunostimulant of a B cell epitope or a synthetic peptide hapten and use it for preparing a vaccine and Antibodies and developmental drugs and detection reagents, preparation of vaccines and antibodies and derivatives and modifications for the treatment of high Th1 diseases including but not limited to type 1 diabetes and its complications, atherosclerosis, cardiovascular disease, rheumatoid joints Diseases such as inflammation and senile dementia and detection of specific target antigens.
  • Th epitope T helper epitope
  • a second object of the present invention is to provide a technical solution for the preparation of the polypeptide.
  • the present invention relates to a stimulating polypeptide, characterized in that the polypeptide comprises a polypeptide sequence comprising amino acid sequence 1 and amino acid sequence 2, which is the B cell epitope IA2 of the insulinoma associated protein IA-2 ( 5)
  • the amino acid sequence is shown in SEQ ID NO. 1 respectively;
  • the amino acid sequence 2 is the C2 terminal Th2 cell epitope P2 of P277, and the amino acid sequence is shown in SEQ ID NO.
  • the invention provides a method for preparing a polypeptide, characterized in that the determination of the amino acid sequence is carried out according to the following steps:
  • the stimulating polypeptide is formed by directing the N-terminus of the IA2(5) peptide FEYQD and the N-terminus of the Th2 epitope P2 peptide directly or via a flexible peptide or a linker peptide, and is named IA2(5)-P2, IA2(5) -P2-1 (abbreviated IP), IA2(5)-P2-2 (abbreviated 2-IP), IA2(5)-P2-3 (abbreviated 3-IP), etc. Further, the stimulation polypeptide IA2(5)-P2 or IA2(5)-P2-1, IA2(5)-P2-2 or IA2(5)-P2-3 is reconciled with a B cell epitope or a synthetic peptide half.
  • the C-terminus of the antigen (indicated by B) is covalently linked by a flexible peptide or a linker peptide to form a polypeptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1 (abbreviated B-IP), B - IA2(5)-P2-2 (abbreviated B-2-IP), B-IA2(5)-P2-3 (abbreviated B-3-IP), etc., IA2(5)-P2, IA2(5)-
  • the amino acid sequences of P2-1 and IA2(5)-P2-2 are respectively shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7; the stimulating polypeptide can significantly enhance the B cell epitope or the synthetic peptide immunogen. Immunogenicity.
  • the C-terminal and B cell epitopes of the IA2(5) peptide FEYQD or the peptide hapten may be covalently linked through a flexible peptide or a linker peptide, and then the N-terminus of the P2 peptide of the Th2 epitope may be passed through a flexible peptide or Even
  • the ligation of peptides forms the polypeptide immunogen IA2(5)-B-P2, IA2(5)-B-P2-1, IA2(5)-B-P2-2, IA2(5)-B-P2-3 Etc., the immunogenicity of the B cell epitope or the synthetic peptide immunogen B can also be significantly enhanced.
  • the amino acid sequences of IA2(5)-D41-P2, IA2(5)-D41-P2-1 and IA2(5)-O-P2 are shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively. .
  • an amino acid sequence of a flexible peptide or a linker peptide capable of linking a linear B cell epitope IA2 (5) with a P2 peptide to form a stimulating polypeptide, or a C-terminal and B cell of the IA2 (5) peptide FEYQD.
  • the epitope or peptide hapten B is covalently linked to the amino acid sequence of a flexible peptide or a linker peptide joined to the N-terminus of the P2 peptide of the Th2 epitope.
  • the flexible peptide or linker linker is Xaa1Xaa2Xaa3------Xaan, Xaa1Xaa2Xaa3Xaa4Xaa5, Xaa1Xaa2Xaa3Xaa4, Xaa1Xaa2Xaa3, Xaa1Xaa2, Xaa, GGGGG, GGGG, GGG, GGS, GG, G, K, KK or other short peptide.
  • Xaa1Xaa2Xaa3------Xaan, Xaa1Xaa2Xaa3Xaa4Xaa5 and Xaa1Xaa2Xaa3Xaa4 are shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.
  • Xaan is any amino acid residue and n is 1,2,3---any Arabic number, preferably 1-20.
  • a third object of the present invention is to provide a B cell epitope or a hapten peptide of a target antigen which can be linked to the stimulating polypeptide via a flexible peptide or a linker peptide, and the stimulating polypeptide can serve as a B cell epitope or a peptide hapten (B) An immunostimulant that enhances immunogenicity.
  • the preparation method is the stimulation polypeptide IA2(5)-P2, IA2(5)-P2-1 (abbreviated IP), IA2(5)-P2-2 (abbreviation 2-IP), IA2(5)-P2
  • the N-terminus of -3 (abbreviated 3-IP) is covalently linked to the B-cell epitope of the target antigen or the C-terminus of the synthetic peptide immunogen (B) via a flexible peptide or a linker peptide to form a polypeptide immunogen B-IA2 (5) -P2, B-IA2(5)-P2-1 (abbreviated B-IP), B-IA2(5)-P2-2 (abbreviated B-2-IP), B-IA2(5)-P2-3( Shorthand B-3-IP) and so on.
  • D41 is a segment of the specific polypeptide of DPP4, 88VFLENSTFDE97, with a total of 10 amino acid residues, and the amino acid sequence is shown in SEQ ID NO. O is yellow
  • a specific polypeptide of ⁇ oxidoreductase, 1101RLEPYKKKNPS1111, has 11 amino acid residues, and the amino acid sequences are shown in SEQ ID NO. H and H1 are a specific polypeptide of xanthine oxidoreductase, and the amino acid sequences are shown in SEQ ID NO. 13 and SEQ ID NO.
  • D41-IP amino acid sequence is shown in SEQ ID NO. 15
  • D41-2-IP amino acid sequence is shown in SEQ ID NO.
  • a fourth object of the present invention is to provide the stimulating polypeptide and the polypeptide immunogen comprising the stimulating polypeptide, which can also be used in recombinant microorganisms and transgenic animals or plants and cells thereof, and can be used as a production plant to produce a polypeptide containing the stimulating polypeptide.
  • a fifth object of the present invention is to provide a composition and a tandem comprising an immunologically effective amount of the polypeptide immunogen comprising the stimulatory polypeptide and a coupled protein thereof, an antigenic peptide of the fusion protein, and a protein.
  • a pharmaceutically acceptable carrier or a pharmaceutically acceptable adjuvant is also included.
  • the pharmaceutically acceptable pharmaceutically active substance including but not limited to: insulin, biguanide, sulfonylurea; wherein the biguanide includes phenformin, butyl hydrazine, metformin; sulfonylureas including phenylurea, rice Ke pancreatic, gliclazide, dextran, glibenclamide, gliclazone, glucosamine, mepyridine, sugar and kidney equality.
  • pharmaceutically acceptable carriers include, but are not limited to, heat shock protein HSP60/65, asparaginase, asparaginase-TTP, asparaginase-TTP-CETPC, Fc, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), polyethylene glycol, and the like.
  • the pharmaceutically acceptable adjuvant is a filler, a diluent, an excipient, an adjuvant, or the like.
  • a filler for example, but not limited to: Freund's adjuvant, aluminum adjuvant, lactose, sucrose, glucose, starch, cellulose (such as carboxymethyl cellulose, hydroxypropylmethyl cellulose, etc.), ethylene glycol, soybean oil, sesame oil , ethanol, sterile saline, sterile water, mannitol plus Lipofundin force Bao Ning and so on.
  • excipients are fillers, diluents, excipients and the like.
  • lactose sucrose, glucose, starch, cellulose (such as carboxymethylcellulose, hydroxypropylmethylcellulose, etc.), ethylene glycol, soybean oil, sesame oil, ethanol, sterile saline, no Bacterial water, etc.
  • the polypeptide immunizing animal containing the stimulating polypeptide can significantly improve the B cell epitope of the target antigenic site or the immunogenicity of the synthetic hapten and significantly increase the amount of the Th2 subtype antibody, and the polypeptide immunogen containing the stimulating polypeptide is used for Preparation of a specific antibody that binds to a target antigen protein containing a B cell epitope or a peptide hapten can reduce the content and/or activity of the target protein, significantly regulate the Th1/Th2 balance disorder, shift from Th1 to Th2, and significantly improve the disease. Symptoms of the disease.
  • a pharmaceutical preparation comprising the stimulating polypeptide and derivatives and analogs thereof, and the coupled protein and fusion protein and immunospecific specific antibodies and antibody derivatives and analogs thereof, and the stimulating polypeptide and the polypeptide containing the stimulating polypeptide
  • the immunogen is used in recombinant microorganisms and transgenic animals or plants and their cells, as a production plant, to produce polypeptide immunogens and fusion proteins and coupled proteins containing the stimulatory polypeptide or to make oral vaccines or to produce specific antibodies and derivatives thereof and
  • a pharmaceutical preparation of an analogue for preventing and treating Th1 dominant disease-induced rheumatism, multiple sclerosis, diabetes, kidney disease, atherosclerosis, hyperlipemia, hyperuricemia, senile dementia, eye disease, podiatry, cardiovascular, etc. It has important application value, and the specific antibodies and their derivatives and analogs or modifications produced can be used for controlling related diseases or detecting related antigenic proteins, and are developed as drugs or detection reagents.
  • the B cell epitope or synthetic hapten is selected as the dominant B cell epitope or synthetic peptide hapten of human dipeptidyl peptidase 4 (DPP4), it is passed through the flexible peptide with the N-terminus of the stimulating polypeptide.
  • DPP4 human dipeptidyl peptidase 4
  • the linker peptide is ligated to form the polypeptide immunogen D41-IP (D41-IA2(5)-P2-1), D41-2-IP and D41-3-IP or it is linked between IA2(5) and P2 to form Peptide immunogens such as IA2(5)-D41-P2-1, which produce high levels of antibodies after immunization of normal mice, but do not cause hypoglycemia, are safe; can produce specificity in mice immunized with diabetes DPP4-bound antibody (in the Th2 subtype), elevated Th2 cytokines, regulates Th1/Th2 balance, increases GLP-1 levels, increases insulin levels, protects islet cells, lowers blood glucose, and can treat abnormalities with DPP4, Provides new ways for diseases such as diabetes, kidney disease, atherosclerosis, senile dementia, eye disease, foot disease, cardiovascular disease caused by abnormal blood glucose, disorder of blood sugar metabolism and Th1/Th2 imbalance; peptide vaccine containing DPP4 dominant B cell epitope Develop diabetes prevention, including type 1 diabetes, type 2
  • the B cell epitope or synthetic hapten when selected as the dominant B cell epitope or synthetic peptide hapten of human xanhtine oxidoreductase (XOD), it is passed through the flexible peptide or the N-terminus of the stimulating polypeptide or The linker peptides are joined to form a polypeptide immunogen O-IA2(5)-P2, O-IA2(5)-P2-1, O2-IA2(5)-P2-2 and other polypeptide immunogens, or are ligated to IA2 ( 5) In the middle of P2, IA2(5)-O-P2 is formed, which can produce specific antibodies binding to xanthine oxidase (belonging to Th2 subtype) after immunizing mice, raising Th2 cytokines and regulating Th1 /Th2 balance, increase insulin volume, protect islet cells, lower blood sugar, reduce xanthine oxidase activity, and increase antioxidant capacity.
  • XOD human xanhtine oxidoreducta
  • Vaccines and antibodies to treat diabetic complications can be developed.
  • the monoclonal antibody prepared by immunizing the animal with the polypeptide immunogen belongs to the Th2 subtype, and is administered to the model rats with high uric acid.
  • the blood glucose of the animal is not lowered, and the specific activity can be combined with xanthine oxidase to increase the Th2 class.
  • Cytokines, regulate Th1/Th2 balance, reduce xanthine oxidase activity, reduce serum uric acid and creatinine, increase urine Acid and creatinine are excreted to protect the kidneys. It can be developed for the preparation of antibodies and Th2 antibodies for the treatment of related diseases or for detection.
  • the B cell epitope or synthetic hapten when selected as the dominant B cell epitope or synthetic peptide hapten of human HMG-CoA reductase (the rate-limiting enzyme in the process of hepatocyte synthesis of cholesterol), it is combined with a stimulating polypeptide.
  • the N-terminus is linked by a flexible peptide or a linker peptide to form a peptide immunogen such as HIP1 and H24IP1, which can produce a specific antibody that binds to HMG-CoA reductase (belongs to the Th2 subtype) and reduces HMG.
  • -CoA reductase enzyme activity, lowering blood lipids, lowering uric acid, raising SOD, etc. can be developed for the preparation of antibodies and Th2 antibodies for the treatment of diseases or detection.
  • the B cell epitope or peptide hapten is selected as the dominant B cell epitope of human uric acid transporter 1 (URAT1), it is linked to the N-terminus of the stimulating polypeptide via a flexible peptide or a linker to form a polypeptide immunogen UIP- 1, after immunization of mice can produce specific antibodies that bind to URAT1 (belonging to the Th2 subtype), lower blood sugar, increase uric acid excretion, lower serum uric acid and creatinine, and protect the kidneys. It can be developed for the preparation of antibodies and Th2 antibodies for the treatment of related diseases or detection, has important theoretical value and broad application prospects, and has important social and economic benefits.
  • a target antigenic protein such as rheumatoid arthritis, senile dementia, etc., a dominant B-cell epitope or synthetic peptide hapten of a Th1/Th2 imbalance-related disease in which human Th1 predominates
  • the peptide or the linker peptide is linked to form a polypeptide immunogen, which, after immunizing the mouse, produces an antibody that binds specifically to a key antigenic protein containing a B cell epitope (which is dominant in the Th2 subtype) and improves disease symptoms. It can be developed for the preparation of antibodies and Th2 antibodies for the treatment of related diseases or for detection.
  • the target antigenic protein of other related diseases When the target antigenic protein of other related diseases is selected, it is linked to the N-terminus of the stimulating polypeptide through a flexible peptide or a linker peptide to form a polypeptide immunogen, which can produce a specific and B-cell epitope-containing epitope after immunization of the mouse.
  • Antigen-protein-bound antibodies are available for the treatment and development of detection reagents.
  • the dominant B cell epitope or synthetic peptide hapten When selecting other dominant antigenic proteins, the dominant B cell epitope or synthetic peptide hapten will The N-terminus of the stimulating polypeptide is linked to the polypeptide immunogen by a flexible peptide or a linker peptide, and after immunizing the mouse, a specific antibody that binds to a key antigenic protein containing a B cell epitope is produced ( belonging to the Th2 subtype).
  • Advantages can be developed for the preparation of a variety of antibodies and Th2 antibodies for detection. It has important theoretical value and broad application prospects, and has important social and economic benefits.
  • Figure 1 is a polypeptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1) (abbreviated B-IP) containing the stimulatory polypeptide of the present invention, B-IA2(5)-P2-2 (B-2-IP), dominant B cell epitopes or peptides of IA2(5)-B-P2-1 (B are human DPP4, xanthine oxidase, HMG-CoA reductase and urate transporter 1 respectively)
  • the hapten constitutes the polypeptide immunogen D41-IP, D41-2-IP, O-IA2(5)-P2, O-IA2(5)-P2-1, IA2(5)-O-P2, H24IP1, HIP1.
  • UIP-1) ELISA and potency test results of specific antibodies produced by serum from C57BL/6J male rats;
  • B-IA2(5)-P2 is a polypeptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1 (abbreviated B-IP) containing the stimulating polypeptide of the present invention, and B-IA2(5)-P2-2 abbreviated B-2-IP), IA2(5)-B-P2-1, etc.
  • B is human DPP4, xanthine oxidase, HMG-CoA reductase and urate transporter 1 dominant B cell epitope or peptide hapten
  • the ELISA assay results of the specific antibody subtypes produced by the serum of the C57BL/6J male mice immunized with the polypeptide immunogens D41-IP, O-IA2(5)-P2-1, HIP1 and UIP-1, respectively;
  • Figure 3 shows the polypeptide immunogen D41-, which is the dominant B cell epitope or peptide hapten of the polypeptide immunogen B-IA2(5)-P2-1 (B is human DPP4, xanthine oxidase, respectively) containing the stimulatory polypeptide.
  • D41- the polypeptide immunogen
  • B-IA2(5)-P2-1 B is human DPP4, xanthine oxidase, respectively
  • Figure 4 is a polypeptide immunogen of the polypeptide B-IA2(5)-P2-1 (B, respectively, human DPP4, xanthine oxidase) having a stimulating polypeptide, which is a dominant B cell epitope or a peptide hapten, respectively.
  • D41-IP O-IA2(5)-P2-1
  • the specific antibodies produced by sera from C57BL/6J male mice were compared with human DPP4 (Fig. 4A, Lane 1: marker; Lane 2: DPP4; Lane 3: D41).
  • -BSA; Lane 4: BSA; Lane 5: L-ASP-independent protein) and human xanthine oxidase Fig.
  • Figure 5 is a diagram showing the dominant B cell epitope or peptide hapten of the polypeptide-containing polypeptide immunogen B-IA2(5)-P2-1 (B is human DPP4, xanthine oxidase, HMG-CoA reductase, respectively)
  • B is human DPP4, xanthine oxidase, HMG-CoA reductase, respectively
  • the polypeptide immunogens D41-IP, D41-2-IP, O-IA2(5)-P2, O-IA2(5)-P2-1, IA2(5)-O-P2, HIP1 and H24IP1 were respectively constructed to immunize C57BL. /6J male mice inhibited the results of human DPP4 (Fig. 5A), oxidase (Fig. 5B) and HMG-CoA reductase activity (Fig. 5C);
  • Figure 6 is a polypeptide containing the stimulating polypeptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1, B-IA2(5)-P2-2, IA2(5)-B-
  • the dominant B cell epitope or peptide hapten of P2-1 (B is human DPP4, xanthine oxidase, HMG-CoA reductase and urate transporter 1 respectively) constitutes the polypeptide immunogen D41-IP, O-IA2 (5 )-P2-1 and UIP-1 (Fig. 6A), HIP1 and H24IP1 (Fig. 6B) immunoblotting results after immunization of C57BL/6J male rats;
  • Figure 7 is a polypeptide-containing immunogen B-IA2(5)-P2-1 (B is a dominant B cell epitope or a peptide hapten of human DPP4, xanthine oxidase, respectively, which constitutes a polypeptide immunogen D41- IP, O-IA2(5)-P2-1 immunized C57BL/6J male rats were tested for GLP-1 by serum ELISA;
  • Figure 8 is a diagram showing the polypeptide-containing polypeptide immunogen B-IA2(5)-P2-1 (B is the dominant B cell epitope or peptide hapten of DPP4, xanthine oxidase, HMG-CoA reductase, respectively)
  • B is the dominant B cell epitope or peptide hapten of DPP4, xanthine oxidase, HMG-CoA reductase, respectively
  • the peptide immunogens D41-IP, O-IA2(5)-P2-1, UIP-1, HIP1, H24IP1 were used to immunize C57BL/6J male rats with blood lipids, uric acid and creatinine;
  • Figure 9 is a polypeptide-containing polypeptide immunogen B-IA2(5)-P2-1 (B is a dominant B cell epitope or a peptide hapten of HMG-CoA reductase, respectively, which constitutes a polypeptide Immunogens O-IA2(5)-P2-1, UIP-1, HIP1, H24IP1) were used to immunize C57BL/6J male rats with anti-oxidative stress indicators GSH-PX, CAT, SOD, MDA detection results;
  • FIG. 10 is a diagram showing the dominant B cell epitope of the polypeptide-containing polypeptide immunogen B-IA2(5)-P2-1 (B is human DPP4, xanthine oxidase, HMG-CoA reductase and uric acid transporter 1 respectively).
  • B is human DPP4, xanthine oxidase, HMG-CoA reductase and uric acid transporter 1 respectively.
  • peptide hapten constitutes the polypeptide immunogen D41-IP (Fig. 10C), O-IA2(5)-P2-1 (Fig. 10D), HIP1 (Fig. 10E) and UIP-1 (Fig. 10F), respectively, to immunize C57BL/6J.
  • Figure 10A is the solvent
  • Figure 10B is the IA2(5)-P2-1 group;
  • Example 1 A polypeptide containing a T helper epitope (Th epitope) IA2(5)-P2, IA2(5)-P2-1 (abbreviated IP), IA2(5)-P2-2 (abbreviation 2 Preparation and synthesis of -IP) and IA2(5)-B-P2-1.
  • Thi epitope T helper epitope
  • IA2(5)-P2-1 abbreviated IP
  • IA2(5)-P2-2 abbreviation 2 Preparation and synthesis of -IP
  • IA2(5)-B-P2-1 A polypeptide containing a T helper epitope (Th epitope) IA2(5)-P2, IA2(5)-P2-1 (abbreviated IP), IA2(5)-P2-2 (abbreviation 2 Preparation and synthesis of -IP) and IA2(5)-B-P2-1.
  • the peptide or linker peptide amino acid sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • IA2(5)-P2, IA2(5)-P2-1IA2(5)-P2-2 amino acid sequences are shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 respectively; or IA2 (5)
  • the C-terminus of the peptide FEYQD and the B-cell epitope or peptide hapten are covalently linked by a flexible peptide or a linker peptide, and then form a polypeptide immunogen by ligation of a flexible peptide or a linker peptide at the N-terminus of the P2 peptide of the Th2 epitope.
  • IA2(5)-B-P2, IA2(5)-B-P2-1, etc., IA2(5)-D41-P2, IA2(5)-D41-P2-1 and IA2(5)-O-P2 Amino acid sequence See also SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. The peptide was synthesized by FMOC solid phase synthesis, and the purity was >90% by HPLC.
  • Example 2 Preparation and synthesis of B cell epitopes or synthetic peptide haptens B.
  • B cell epitope analysis software and online analysis tools based on single parameter, combined with secondary structure prediction and spatial structure characteristics, respectively, from a target antigen protein (such as: DPP4 or XOD or HMG-CoA reductase and uric acid transport
  • a target antigen protein such as: DPP4 or XOD or HMG-CoA reductase and uric acid transport
  • Multiple dominant B cell epitopes or peptide haptens B are selected from more than 100 epitopes of protein 1), by IA2(5)-P2, IA2(5)-P2-1 (abbreviated IP), IA2 ( 5)-B-P2-1, etc. are linked by a flexible peptide, and any process of re-engineering and screening is screened by an immunized animal experiment to obtain an immunogenic B cell epitope or a synthetic peptide hapten B.
  • the peptides were synthesized by FMOC solid phase synthesis, and the purity of HPLC was >90%.
  • Example 2-2 When B is the dominant B cell epitope of human xanhtine oxidoreductase (XOD), O1, O2, O3----- and so on are selected from more than 100 epitopes.
  • a dominant B cell epitope or peptide hapten B with IA2(5)-P2, IA2(5)-P2-1 (abbreviated IP), IA2(5)-P2-2 (abbreviated 2-IP),
  • the N-terminus of IA2(5)-B-P2 is linked by a flexible peptide to form a peptide immunogen, and any process of re-engineering and screening is screened by an immunized animal experiment to obtain a highly immunogenic B cell epitope or peptide hapten O, O1 and so on.
  • O is a specific polypeptide of xanthine oxidoreductase 1101RLEPYKKKNPS1111, a total of 11 amino acid residues, the amino acid sequence is shown in SEQ ID NO.
  • H and H1 are a specific polypeptide of xanthine oxidoreductase, and the amino acid sequences are shown in SEQ ID NO. 13 and SEQ. ID NO.14.
  • Example 2-5 When B is the dominant B cell epitope of a key antigenic protein of a Th1/Th2 imbalance-associated disease in which Th1 predominates, B1, B2, B3---- And so on, multiple dominant B cell epitopes or synthetic peptide haptens B, linked by flexible peptides to the stimulating peptides IA2(5)-P2, IA2(5)-P2-1, IA2(5)-P2-2, etc.
  • the peptide immunogen is formed, and the immunogenic animal B-epitope epitope or peptide hapten B1, B2 is obtained by immunological animal experiment, which is synthesized by FMOC solid phase synthesis method, and the purity of HPLC is >90%.
  • Example 3 Peptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1 (abbreviated B-IP), B-IA2(5)-P2-2 (abbreviated B-2-IP) (B is a B cell epitope or a synthetic peptide hapten) preparation and synthesis.
  • B-cell epitope analysis software and online analysis tools based on multiple single parameters, combined with secondary structure prediction and spatial structure characteristics, from a target antigen protein (eg DPP4 or Select more than one dominant B cell epitope or peptide hapten B from more than 100 epitopes of XOD or HMG-CoA reductase and urate transporter 1, etc., and the C-terminus of B and IA2(5)-P2 N-terminal connection of -1, constitutes B-IA2(5)-P2, B-IA2(5)-P2-1, B-IA2(5)-P2-2, IA2(5)-B-P2, IA2( 5)-P-P2-1 and other polypeptide immunogens, through software analysis and animal experiments, multiple screening and transformation, to obtain high-immunity containing stimulating polypeptide IA2(5)-P2, IA2(5)-P2- 1 (abbreviated IP), IA2(5)-P2-2 (abbreviated 2-IP) peptide immunogen B-IA2
  • D41-2-IP amino acid sequence see SEQ ID NO. 16
  • O-IA2(5)-P2 amino acid sequence see SEQ ID NO. 17
  • O-IA2 (5)-P2-1 amino acid sequence is shown in SEQ ID NO. 18
  • IA2 (5)-O-P2-1 amino acid sequence is shown in SEQ ID NO. 18
  • HIP1 amino acid sequence is shown in SEQ ID) NO. 19
  • H24IP1 amino acid sequence see SEQ ID NO. 20
  • UIP-1 amino acid sequence see SEQ ID NO.
  • polypeptides described above were synthesized by FMOC solid phase synthesis, and the purity was >90% by HPLC.
  • the peptides for analysis were synthesized by FMOC solid phase synthesis, and the purity was >95% by HPLC, and then coupled with protein BSA or KLH.
  • Example 4 Polypeptide immunogen B-IA2(5)-P2, B-IA2(5)-P2-1, B-IA2(5)-P2-2, IA2(5)-B containing the stimulatory polypeptide of the present invention -P2, IA2(5)-B-P2-1 (B is the dominant B cell epitope or peptide hapten of human DPP4, xanthine oxidase, HMG-CoA reductase and uric acid transporter 1, respectively) D41-IP, D41 Immunogenicity and pharmacodynamics of -2-IP, O-IA2(5)-P2, IA2(5)-O-P2, O-IA2(5)-P2-1, H24IP, HIP1 and UIP-1) Evaluation.
  • Example 4-1 the polypeptide immunogen B-IA2(5)-P2 containing the stimulating polypeptide of the present invention, B-IA2(5)-P2-1, B-IA2(5)-P2-2, IA2(5)-B-P2, IA2(5)-B-P2-1 (B is human DPP4, xanthine oxidation, respectively
  • B is human DPP4, xanthine oxidation, respectively
  • the dominant B cell epitope or peptide hapten of the enzyme, HMG-CoA reductase and uric acid transporter 1 constitute the polypeptide immunogen D41-IP, D41-2-IP, O-IA2(5)-P2, IA2 (5, respectively) -O-P2, O-IA2(5)-P2-1, H24IP, HIP1 and UIP-1) Immunogenicity and pharmacodynamic evaluation of STZ-induced diabetic model rats.
  • a mouse model of diabetes mellitus (DM) was induced by sequential intraperitoneal injection of streptozotocin (STZ) in small doses.
  • STZ streptozotocin
  • 4-week-old male C57BL/6J mice purchased from the Comparative Medical Center of Yangzhou University, license number: SCXR (Su) 2012-0004.
  • the STZ concentration was 7.5 mg/ml with 0.1 M pH 4.4 citrate buffer.
  • blood glucose values were measured on the 3rd, 7th and 14th day after modeling, and the fasting blood glucose was ⁇ 11.1mmol/L twice as the standard.
  • Example 4-1-2 Treatment experiment of animal immunity after successful modeling
  • More than 100 diabetic mice were randomly divided into 13 groups, 10 in each group: placebo group (solvent control group, negative control group), IA2(5)-P2, IA2(5)-P2-1, IA2 ( 5)-P2-2 control group, D41-IP, D41-2-IP, O-IA2(5)-P2, IA2(5)-O-P2, O-IA2(5)-P2-1, H24IP, HIP1 and UIP-1 administration groups.
  • the drug concentration in the drug-administered group was 1 mg/mL, and the dose was 100 ⁇ g/time/time, that is, 100 ⁇ L of the drug solution; the negative control group: 100 ⁇ l of the oil agent/only/time.
  • the drug preparation method temporarily disposes the oil agent in a ratio of 1 mg + 40 mg of mannitol dissolved in 1 ml of 20% Lipofundin. Animal immunization was started at 2 weeks after the model was successful (with blood glucose ⁇ 11.1 mmol/L). The drug was administered once a week for 5 times, and then immunized three times at 7, 9, and 11 weeks, for a total of 8 times.
  • Examples 4-1-3 Polypeptide immunogens containing the stimulating polypeptide of the present invention D41-IP, D4-2-IP, IA2(5)-D41-P2-1, IA2(5)-D41-P2, O-IA2 (5) ELISA and specific antibodies produced by sera from C57BL/6J male mice immunized with -P2, IA2(5)-O-P2, O-IA2(5)-P2-1, H24IP, HIP1 and UIP-1, respectively Resistance Body price test:
  • Dilute X-BSA with a dilution solution 0.05 M sodium carbonate-sodium bicarbonate buffer, pH 9.6
  • BSA 0.05 M sodium carbonate-sodium bicarbonate buffer, pH 9.6
  • the serum antibody titers of D41-IP, D41-2-IP, IA2(5)-D41-P2-1, O-IA2(5)-P2-1, HIP1, H24IP1 and UIP-1 are all between 8,000 and 20,000. Between (see Table 1), it can be used to prepare antibodies, develop therapeutic drugs and detection reagents.
  • Example 4-1-4 Specificity of the polypeptide-containing polypeptide immunogens D41-IP, O-IA2(5)-P2-1, HIP1 and UIP-1 of the present invention, respectively, after immunization of C57BL/6J male rats ELISA detection of antibody subtypes:
  • Example 4-1-5 Cytokine assay for the polypeptide immunogen D41-IP and O-IA2(5)-P2-1 containing the stimulatory polypeptide of the present invention after immunotherapy STZ-induced diabetic C57BL/6J male model Measurement:
  • Cytokine assays were performed according to the kit instructions. The brief experimental procedure is 1. Dilution and loading of standards and samples. Accurately add 50 ⁇ L of standard, or 5-fold diluted serum, to a 96-well plate. 2. Incubate, seal with a sealing plate and incubate at 37 ° C for 30 minutes. 3. Wash, discard the liquid and dry it. Fill each well with the washing solution, let stand for 30 s and discard it. Repeat this 5 times and pat dry. 4. Add 50 ⁇ l of enzyme labeling reagent to each well, except for blank wells. 5, incubation, operation the same as 2. 6, washing, the same operation as 3. 7.
  • D41-IP and O-IA2(5)-P2-1 can regulate Th1/Th2 imbalance in diabetic mice, and more intervention immune balance to Th2 transformation.
  • Immunization with D41-IP, O-IA2(5)-P2-1 can be used to treat diseases caused by Th1/Th2 imbalance in the body, such as type 1 diabetes and its complications, inflammatory reactions and other diseases associated with Th1/Th2 imbalance.
  • Example 4-1-5 Polypeptide vaccine containing stimulating polypeptide D41-IP, O-IA2(5)-P2-1 were respectively incubated with C57BL/6J male mice, and the specific antibodies produced by the serum were oxidized with human DPP4 and xanthine, respectively.
  • DPP4 was transferred to a PVDF membrane according to the standard operating procedure of Western Blot, and the transfer conditions were 100 V, 5 h; the transferred PVDF membrane was shaken for 1 h at room temperature in 25 mL of blocking buffer. Wash 15 mL TBST 3 times (5 min / time); then immerse the PVDF membrane in the 1:10 diluted antigen peptide-immunized mouse serum, incubate for 1-2 h or 4 ° C overnight at room temperature, shake slowly, wash 3 times with 15 mL TBST (5 min / time). A 1:5000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG was added and incubated for 1 h at 37 °C.
  • B-IA2(5)-P2-1 including D41-IP, O-IA2(5)-P2-1
  • the target protein antigen D of B or the xanthine oxidase and the major fragment specifically bind, and do not bind to an unrelated protein, and the antibody can be developed for detecting the target antigen and its fragment.
  • Example 4-1-6 Stimulating polypeptide-containing polypeptide immunogen D41-IP, O-IA2(5)-P2-1, HIP1 and H24IP1, respectively, immunized with C57BL/6J male rats to inhibit DPP4 and xanthine oxidation in vitro.
  • Enzyme activity and serum HMG-CoA reductase assay :
  • Example 4-1-6-1 Peptide immunogen immunization C57BL/6J male rats inhibited the inhibition rate of in vitro enzymatic activity.
  • the total reaction system was 100ul, serum, buffer, The enzyme was incubated at 37 ° C for 30 minutes; 4 groups were added to the 96-well plate in the order of enzyme, buffer and substrate, and reacted in a 37 ° C incubator for 60 min to measure the absorbance at 405 nm. The values of OD405 for each group were compared and the inhibition rate was calculated. The results are shown in Fig. 5A. The results showed that the serum produced by D41-IP immunized mice significantly inhibited the activity of DPP4 in vitro compared with the solvent and IA-2(5)-P2-1 (P ⁇ 0.001).
  • Example 4-1-6-2 ELISA of serum HMG-CoA reductase activity after immunization of C57BL/6J male mice by HIP1
  • the kit was purchased from Shanghai Baoman Co., Ltd., and the measurement method was carried out according to the instructions. The results are shown in Fig. 5C. The results showed that compared with the solvent and IA-2(5)-P2-1, the activity of HMG-CoA reductase in the serum produced by HIP1 immunized mice was significantly decreased (P ⁇ 0.01), and H24IP1 immunization was performed. The activity of HMG-CoA reductase in serum produced by mice was significantly decreased (P ⁇ 0.05).
  • Examples 4-1-7-1 Peptide-containing peptide immunogens D41-IP, D41-2-IP, IA2(5)-D41-P2-1, O-IA-2(5)-P2-1 , H24IP, HIP1 and UIP-1 were used to immunize C57BL/6J male mice after hypoglycemic experiments:
  • Example 4-1-7-2 Detection of hypoglycemia and mortality and morbidity of a peptide immunogen D41-IP containing a stimulating peptide after immunological control of a NOD mouse type 1 diabetes model:
  • 3 groups were randomly divided into 10 groups: solvent group (negative control group), P277 group (positive control group), and D41-IP group. Administered according to 7, 19, 20, 21, 22, 23, 25, 27, 29. Multiple immunizations under the skin, immunization of the limbs and the back.
  • the drug concentration in the drug-administered group was 1 mg/mL, and the dose was 100 ⁇ g/time/time.
  • the negative control group 100 mL oil agent was used to protect the body.
  • the blood glucose of the mouse was periodically measured by a blood glucose meter using a method of blood withdrawal.
  • the blood glucose changes, morbidity and mortality of the mice after administration of the immunogen containing the carrier peptide peptide in NOD female mice are shown in Table 2.
  • the results showed that the peptide immunogen D41-IP could significantly control the blood glucose, morbidity and mortality of NOD mice compared with the control group.
  • the blood glucose of the mice returned to normal, with no morbidity and mortality.
  • Example 4-1-8 Polypeptide-containing polypeptide immunogen D41-IP, O-IA2(5)-P2-1 were respectively immunized with C57BL/6J male rats, and serum ELISA was used to detect GLP-1 or insulin levels:
  • the determination of GLP-1 levels in mouse serum was performed according to the kit instructions (Enzyme Biotech Co., Ltd.). The OD value was measured at a wavelength of 450 nm according to the kit instructions. After D41-IP and O-IA2(5)-P2-1 were immunized with C57BL/6J male rats for 7 times, serum GLP-1 levels were measured. The experimental results are shown in Figure 7. The results showed that the level of GLP-1 in mice was significantly increased after D41-IP immunization, and there was statistical difference between the solvent group and IA2(5)-P2-1 (P ⁇ 0.01). After O-IA2(5)-P2-1 immunization, insulin levels in mice increased significantly, compared with the solvent group (P ⁇ 0.05). Can develop treatment diabetes.
  • Example 4-1-9 Polypeptide-containing polypeptide immunogen O-IA2(5)-P2-1 and HIP1 and UIP-1 immunized C57BL/6J male rats after blood lipid, uric acid, creatinine and anti-oxidative stress indicators Detection
  • the detection reagents for serum lipid, uric acid and anti-oxidative stress in mice were provided by Nanjing Jiancheng Co., Ltd., and tested and calculated according to the kit instructions.
  • the experimental results are shown in Fig. 8 and Fig. 9.
  • the experimental results show that after immunization of mice with O-IA2(5)-P2-1 and HIP1 and UIP-1, serum uric acid and creatinine are significantly decreased, and antioxidant activity is significantly enhanced. , with statistical difference (P ⁇ 0.05). After immunization of mice with HIP1, serum lipid levels in mice were significantly decreased (P ⁇ 0.05). Can alleviate diabetic complications.
  • Example 4-1-10 Polypeptide-containing polypeptide immunogen D41-IP, O-IA2(5)-P2-1, HIP1 and UIP-1 immunotherapy Immunohistochemical identification of pancreas in C57BL/6J male rats ;
  • the animal tissues of the experimental group and the control group were taken as fresh as possible, washed with PBS (phosphate buffer), fixed and embedded in tissue pieces less than 0.5 cm ⁇ 0.5 cm ⁇ 0.1 cm, sliced, and insulin was added to the sections.
  • Antibody treatment dropwise addition of biotinylated secondary antibody (IgG), followed by addition of horseradish-labeled streptavidin working solution (SA/HRP), washing at 37 ° C for 20 minutes, 0.1 M PBS for 3 times ⁇ 5 minutes; DAB color development, washing, neutral resin sealing, microscopic observation: 200 ⁇ micrographs of positive and negative tissues of the experimental group and the control group were selected.
  • the experimental results are shown in Figure 10.
  • Example 4-2 The polypeptide immunogen B-IA2(5)-P2-1 containing the stimulatory polypeptide of the present invention (B is a dominant B cell epitope or a peptide hapten of human DPP4, xanthine oxidase, respectively, and constitutes a polypeptide immunization Immunogenicity and pharmacodynamic evaluation of the original D41-IP, O-IA2(5)-P2-1 immune type 1 diabetes NOD model mice.
  • a target antigenic site B cell epitope or peptide hapten
  • for preparing a specific antibody and a Th2 subtype antibody that bind to a target antigen protein containing a B cell epitope or a peptide immunogen A polypeptide immunogen that stimulates a polypeptide, an antibody based on the production thereof, and a specific antibody against a Th2 subtype, and a pharmaceutical preparation containing the polypeptide, a derivative thereof and an analog thereof, and a specific antibody and antibody derivative and the like produced thereby, in the prevention and treatment Th1 is dominant in the detection of diseases and other important applications.
  • Antibodies and Th2 subtype specific antibodies and their derivatives and analogs can detect related target antigen proteins and develop them as detection reagents.
  • B is the dominant B cell epitope of DPP4
  • it produces a specific high titer antibody that binds to DPP4 and a Th2 subtype antibody, inhibits DPP4 activity, increases GLP-1, protects ⁇ cells, increases insulin secretion, and significantly decreases.
  • Blood sugar, lowering uric acid and creatinine can be used for the treatment of DPP4 abnormalities, abnormal blood glucose, disorders of blood glucose metabolism and Th1/Th2 imbalance, diabetes, kidney disease, atherosclerosis, senile dementia, eye disease, podiatric disease, cardiovascular disease, etc. .
  • the prepared antibodies and Th2 subtype antibodies can be developed for therapeutic and detection purposes.
  • B is the dominant B cell epitope of human xanhtine oxidoreductase (XOR)
  • the polypeptide immunogen whose C-terminus is linked to IA2(5)-P2-1 can produce specificity after immunization of mice.
  • An antibody that binds to xanthine oxidase (belongs to the Th2 subtype dominant antibody) raises Th2 cytokines, regulates Th1/Th2 balance, increases insulin levels, protects islet cells, lowers blood glucose, and reduces xanthine oxidase activity , increased the ability to enhance antioxidant capacity.
  • Vaccines and antibodies to treat diabetic complications can be developed.
  • monoclonal antibodies and genetically engineered antibodies can be prepared by hybridoma technology, antibody library technology, transgenic animal technology, single cell technology, etc., which belong to the Th2 subtype, and are administered to treat high uric acid.
  • Model rat Does not reduce animal blood sugar, can specifically bind to xanthine oxidase, increase Th2 cytokines, regulate Th1/Th2 balance, inhibit xanthine oxidase activity, increase insulin secretion, reduce serum uric acid and creatinine, enhance antioxidant Role to protect the kidneys. It can be developed for the preparation of antibodies and Th2 antibodies for the treatment of diseases or detection.
  • B is a human HMG-CoA reductase (a rate-limiting enzyme in the process of hepatocyte synthesis of cholesterol, inhibition of HMG-CoA reductase can block endogenous cholesterol synthesis), a dominant B cell epitope or peptide hapten
  • the polypeptide immunogens HIP1 and H24IP1 linked to IA2(5)-P2-1 at the C-terminus can produce specific antibodies that bind to HMG-CoA reductase (subject to the Th2 subtype dominant antibody) after immunization of mice, reducing HMG-CoA reductase enzyme activity, lower blood lipids, increase insulin secretion, lower blood sugar, enhance anti-oxidation, can be developed for the preparation of antibodies and Th2 antibodies for the treatment of diseases or detection.
  • the polypeptide immunogen UIP-1 which binds its C-terminus to IA2(5)-P2-1, produces specificity in immunized mice.
  • the antibody that binds to URAT1 (which belongs to the Th2 subtype dominant antibody) increases insulin secretion and lowers serum uric acid and creatinine to protect the kidneys. It can be developed for the preparation of antibodies and Th2 antibodies for the treatment of related diseases or for detection.
  • Th1/Th2 imbalance-related diseases in human Th1 such as rheumatoid arthritis, the dominant B cell epitope of Alzheimer's disease, and linking it with a stimulating peptide through a flexible peptide or a linker peptide to form a polypeptide immunization
  • a specific antibody which belongs to the Th2 subtype dominant antibody
  • Th2 antibodies for the treatment of related diseases or for detection.
  • the target antigenic protein of other Th2 dominant Th1/Th2 imbalance-related diseases When selecting the target antigenic protein of other Th2 dominant Th1/Th2 imbalance-related diseases, it is linked with the N-terminus of the stimulating polypeptide through a flexible peptide or a linker peptide to form a polypeptide immunogen, which can produce specificity after immunization of the mouse.
  • An antibody that binds to a key antigen-protein of a B-cell epitope can be used to develop a detection reagent, which has important theoretical value and broad application prospects, and has important social and economic benefits.

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Abstract

本发明公开了一种含T辅助细胞表位（Th表位）的多肽，其可作为目标抗原的B细胞表位或肽半抗原的免疫刺激物。本发明还公开了一种多肽免疫原，其是将目标抗原的B细胞表位或肽半抗原通过连接肽与所述多肽共价连接而成。所述多肽免疫原能显著增强B细胞表位或肽半抗原的免疫原性，制备与所述目标抗原特异性结合的抗体，能显著增强偏向Th2的免疫调节。所述多肽或所述多肽免疫原可用于生产疫苗或特异性抗体，用于防治Th1占优势的疾病，例如糖尿病及其并发症、类风湿性关节炎等，所述多肽或所述多肽免疫原还可用于制备检测试剂。

Description

[根据细则37.2由ISA制定的发明名称]　一种作为B细胞表位或肽半抗原的免疫刺激物的多肽和其构成的多肽免疫原，以及它们的用途 技术领域
本发明涉及一种含T辅助细胞表位(Th表位)的多肽及其应用，尤其是此多肽用于和B细胞表位或肽半抗原---一种目标抗原蛋白的目标抗原位点共价连接成为多肽免疫原，可以作为B细胞表位或肽半抗原的免疫刺激物，增强目标抗原位点(B细胞表位或肽半抗原)的免疫原性并可用于制备特异的与含B细胞表位或肽免疫原的目标抗原蛋白结合的抗体，含有该刺激多肽的多肽免疫原及偶联蛋白和融合蛋白以及基于其生产的特异抗体以及含有该多肽的衍生物和类似物及其基础上产生的特异抗体和抗体衍生物和类似物的药物及制剂，在预防和治疗Th1占优势诱导的疾病等方面具有重要应用价值，特异抗体及其衍生物和类似物可检测相关目标抗原蛋白，开发为检测试剂。本发明涉及免疫学、药学及医学相关领域。
背景技术
人类和动物同样存在两个辅助性T淋巴细胞亚群，Th1细胞主要产生Th1类细胞因子IL-2、TNF-β和IFN-γ；Th2细胞主要产生IL-4、IL-5、IL-6、IL-10和IL-13。正常情况下，辅助性T淋巴细胞亚群Th1/Th2细胞处于平衡状态，但机体发生功能异常时，常表现出平衡偏向其中一方，此即为"Th1/Th2平衡漂移"。习惯上把Th1及其细胞因子占优势的状态称为Th1状态，Th2及其细胞因子占优势的状态称为Th2状态。Th1/Th2平衡影响细胞因子网络的平衡，与许多疾病的发生、发展、治疗、转归有密切的关系，不同的自身免疫性疾病中，Th1/Th2平衡失调的表现各不相同。类风湿性关节炎、1型糖尿病、多发性硬化及慢性甲 状腺炎等疾病中，Th1或Th1分泌的细胞因子占优势,Th1诱导发病，加重病情；Th2则能减轻病症，甚至阻止发病，这与Th1/Th2的炎性及抗炎性反应有关。基于Th1/Th2漂移状态与各种疾病的关系，越来越多的研究正倾向于发现、开发能逆转或稳定Th1/Th2状态的药物和方法。
表位是蛋白质抗原性的基础,蛋白质抗原通过表位体现其免疫特性，就某一蛋白质而言，它含有多种表位如B细胞表位又称抗原决定簇(antigenic determinant，AD)，T辅助细胞(Th)表位等等，还有抑制性表位，毒性表位，交叉反应性表位等等。选择合适的表位并且通过合适的方法与其他肽或者蛋白质等等连接以及用什么佐剂及剂型，如何免疫等等才能得到理想的多肽和蛋白疫苗,产生特异的高效价(高滴度)抗体是有巨大的挑战性的。小鼠体内Th1会产生IgG2a亚型抗体，Th2型则会产生IgG1和IgG2b亚型抗体，人体内Th1会产生IgG1亚型抗体，Th2型则会产生IgG2a亚型抗体，Th2亚型抗体可以增加Th2型细胞因子的产生并能治疗Th1偏高疾病，缓解病症。
当前判断疾病免疫发病机制中Th1或Th2细胞效应为主导的方法，主要是依据炎症组织局部细胞因子的表达，也可根据外周血清中抗体反应亚类的变化进行判断。在动物模型的研究中，根据干扰Th1或Th2细胞效应的各种环节后Th1或Th2细胞效应的变化来判断的与Th1偏高相关的人类感染疾病及自免疫性疾病有1型糖尿病及并发症，肾脏病，肾炎，类风湿性关节炎，反应性关节炎，多发性硬化，自身免疫性甲状腺炎，接触性皮炎，结节性红斑，习惯性流产等等。
B细胞表位或肽半抗原的确定对多肽疫苗的合成、诊断试剂的制备、单克隆抗体的筛选等研究工作具有重要意义，但是B细胞表位或肽半抗原促使机体产生高滴度抗体的前提是要具有强免疫原性，通常为了增加B细胞表位或肽 半抗原的免疫原性，通过将B细胞表位或肽半抗原偶联到KLH、BSA等蛋白或者构建到大分子的融合蛋白上增加Th应答，构建融合蛋白方法存在费时，耗钱，制备周期长等许多不利之处。此外，KLH和BSA免疫后产生的抗体以Th1抗体偏多，用于连接B细胞表位或肽半抗原形成的免疫原是不适合用来治疗Th1偏高的疾病如1型糖尿病及并发症和相关疾病。多肽免疫原诱导特异的抗体应答，必需包括B细胞表位(或肽半抗原)和Th细胞表位，这已经很清楚。选择合适的T辅助细胞(Th)表位及其与B细胞表位或肽半抗原的合适的连接来显著提高B细胞表位或肽半抗原的免疫原性是一个难题。
P277肽是HSP60的437～460位的一段特异性多肽,共24个氨基酸，也是能与效应T细胞反应的抗原决定簇，是在T1DM中发挥作用的特异片段。DiaPep277目前以进入III期临床研究。已有实验研究发现按照国外DiaPep277相同的皮下给药方式，用P277肽进行给药，能引起血管内皮细胞损伤，并诱发肌体产生抗体从而诱发严重的动脉粥样硬化；将P277分成P1肽段(437～450)和P2肽段(448～460)两部分，已经证实P1多肽具有一定的促动脉粥样硬化作用，而P2多肽是P277对T1DM起主要作用的Th2表位肽。
胰岛细胞特异抗原-胰岛素瘤相关蛋白(Insulinoma associated protein-2,IA-2)被认为是启动自身免疫性糖尿病即1型糖尿病(Type 1 diabetes mellitus，T1DM)的主要靶抗原之一，主要在脑部，胰腺，胰岛瘤，神经内分泌细胞中表达。IA-2的近膜区JM2的626～630位的线形序列是一个B细胞表位(626FEYQD630)，命名其为IA2(5)。以IA-2或者其中的B细胞表位免疫模型动物是1型糖尿病防治的一个策略。
发明内容
发明目的：
有鉴于此，本发明的目的之一在于提供一种含T辅助细胞表位(Th表位)的刺激多肽作为B细胞表位或合成肽半抗原的免疫刺激物及将其用于制备疫苗和抗体及开发药物和检测试剂,制备疫苗和抗体及衍生物和改造物可用于治疗Th1偏高疾病包括但不限于1型糖尿病及其并发症、动脉粥样硬化、心血管病、类风湿性关节炎、老年痴呆等疾病及检测相关特异目标抗原。
本发明的目的之二在于提供该多肽制备的技术方案。
本发明涉及一种刺激多肽，其特征在于该多肽含有一个多肽序列，该多肽序列含有氨基酸序列1和氨基酸序列2，所述氨基酸序列1是胰岛素瘤相关蛋白IA-2的B细胞表位IA2(5)，氨基酸序列分别见SEQ ID NO.1；所述氨基酸序列2是P277的C端的Th2细胞表位P2，氨基酸序列分别见SEQ ID NO.2。
在于提供多肽的制备方法，其特征在于其氨基酸序列的确定按照如下步骤实现：
将IA2(5)肽段FEYQD的C端和Th2表位P2肽段的N端直接或通过柔性肽或连接肽段的连接形成该刺激多肽，命名为IA2(5)-P2，IA2(5)-P2-1(简写IP)，IA2(5)-P2-2(简写2-IP)，IA2(5)-P2-3(简写3-IP)等。进一步，将该刺激多肽IA2(5)-P2或IA2(5)-P2-1，IA2(5)-P2-2或IA2(5)-P2-3等再和B细胞表位或合成肽半抗原(以B表示)的C端通过柔性肽或连接肽共价连接，形成多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1(简写B-IP)，B-IA2(5)-P2-2(简写B-2-IP)，B-IA2(5)-P2-3(简写B-3-IP)等，IA2(5)-P2、IA2(5)-P2-1、IA2(5)-P2-2氨基酸序列分别见SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7；该刺激多肽可以显著增强B细胞表位或合成肽免疫原的免疫原性。
进一步，也可以是IA2(5)肽段FEYQD的C端和B细胞表位或肽半抗原通过柔性肽或连接肽共价连接后，再和Th2表位P2肽段的N端通过柔性肽或连 接肽段的连接形成多肽免疫原IA2(5)-B-P2，IA2(5)-B-P2-1，IA2(5)-B-P2-2，IA2(5)-B-P2-3等，也可以显著增强B细胞表位或合成肽免疫原B的免疫原性。IA2(5)-D41-P2，IA2(5)-D41-P2-1和IA2(5)-O-P2的氨基酸序列分别见SEQ ID NO.8，SEQ ID NO.9和SEQ ID NO.10。
进一步，在于提供能将线形B细胞表位IA2(5)与P2肽段连接起来形成刺激多肽的柔性肽或连接肽段的氨基酸序列，或者是IA2(5)肽段FEYQD的C端和B细胞表位或肽半抗原B共价连接再和Th2表位P2肽段的N端连接起来的柔性肽或连接肽段的氨基酸序列。
进一步，所述柔性肽或连接肽接头为Xaa1Xaa2Xaa3------Xaan，Xaa1Xaa2Xaa3Xaa4Xaa5、Xaa1Xaa2Xaa3Xaa4、Xaa1Xaa2Xaa3、Xaa1Xaa2、Xaa、GGGGG、GGGG、GGG、GGS、GG、G、K、KK或其他短肽。Xaa1Xaa2Xaa3------Xaan，Xaa1Xaa2Xaa3Xaa4Xaa5和Xaa1Xaa2Xaa3Xaa4所述氨基酸序列见SEQ ID No.3、SEQ ID No.4和SEQ ID No.5。Xaan为任一种氨基酸残基，n为1,2,3---任何阿拉伯数字，优选1-20数目。
本发明的目的之三在于提供和该刺激多肽能通过柔性肽或连接肽段连接的目标抗原的B细胞表位或半抗原肽，该刺激多肽能作为B细胞表位或肽半抗原(B)的免疫刺激物,增强免疫原性。
进一步，制备方法是将该刺激多肽IA2(5)-P2，IA2(5)-P2-1(简写IP)，IA2(5)-P2-2(简写2-IP)，IA2(5)-P2-3(简写3-IP)的N端通过柔性肽或连接肽和目标抗原的B细胞表位或合成肽免疫原(B)的C端共价连接，形成多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1(简写B-IP)，B-IA2(5)-P2-2(简写B-2-IP)，B-IA2(5)-P2-3(简写B-3-IP)等。D41是DPP4的一段特异性多肽88VFLENSTFDE97,共10个氨基酸残基，氨基酸序列见SEQ ID NO.11。O是黄 嘌呤氧化还原酶的一段特异性多肽1101RLEPYKKKNPS1111,共11个氨基酸残基，氨基酸序列分别见SEQ ID NO.12。H和H1是黄嘌呤氧化还原酶的一段特异性多肽,氨基酸序列见SEQ ID NO.13和SEQ ID NO.14。D41-IP(氨基酸序列见SEQ ID NO.15)、D41-2-IP(氨基酸序列见SEQ ID NO.16)、O-IA2(5)-P2(氨基酸序列见SEQ ID NO.17)、O-IA2(5)-P2-1(氨基酸序列见SEQ ID NO.18)、IA2(5)-O-P2-1、HIP1(氨基酸序列见SEQ ID NO.19)、H24IP1(氨基酸序列见SEQ ID NO.20)。用该多肽免疫原多次小剂量免疫动物可产生高水平的能和B细胞表位或合成肽免疫原特异结合的Th2型占优势抗体。
本发明的目的之四在于提供的该刺激多肽及含该刺激多肽的多肽免疫原也可以用于重组微生物和转基因动物或植物及其细胞，使它们作为生产工厂，能够生产含此刺激多肽的多肽免疫原及其融合蛋白和偶联蛋白或制作口服疫苗或生产特异抗体及其衍生物和类似物，减低目标抗原蛋白的含量和/或活性,显著调节Th1/Th2平衡失调，由Th1偏向Th2转移，显著改善疾病的发病症状。
本发明的目的之五在于提供包含免疫学有效量的所述包含该刺激多肽的多肽免疫原及其偶联蛋白、融合蛋白的抗原肽和蛋白的组合物和串联物。
进一步，还包含药学上可接受的载体或者药学上可接受的辅料。所述的多肽免疫原和多肽疫苗与药物活性物质或药物载体制成组合物后的应用。
所述的药学上可接受的药物活性物质，包括但不限于：胰岛素、双胍类、磺脲类；其中双胍类包括苯乙双胍、丁二胍、二甲双胍；磺脲类药物包括列苯脲、米克胰、格列齐特、克糖利、格列波脲、格列喹酮、糖适平、美吡达、糖肾平等等。
进一步，药学上可接受的载体，包括但不限于：热休克蛋白HSP60/65、门冬酰胺酶、门冬酰胺酶-TTP、门冬酰胺酶-TTP-CETPC，Fc，牛血清白蛋白 (BSA)，钥孔血蓝蛋白(KLH),聚乙二醇等。
进一步，所述药学上可接受的辅料：为填充剂、稀释剂、赋形剂、佐剂等。例如包括但不限于：弗氏佐剂、铝佐剂、乳糖、蔗糖、葡萄糖、淀粉、纤维素类(如羧甲基纤维素、羟丙甲基纤维素等)、乙二醇、豆油、芝麻油、乙醇、无菌生理盐水、无菌水，甘露醇加Lipofundin力保肪宁等。
所述的辅料为填充剂、稀释剂、赋形剂等。例如包括但不限于：乳糖、蔗糖、葡萄糖、淀粉、纤维素类(如羧甲基纤维素、羟丙甲基纤维素等)、乙二醇、豆油、芝麻油、乙醇、无菌生理盐水、无菌水等。
本发明的有益效果在于：
含有该刺激多肽的多肽免疫原免疫动物可以显著改善目标抗原位点的B细胞表位或合成半抗原的免疫原性并显著提高Th2亚型抗体的量，含有该刺激多肽的多肽免疫原用于制备特异的与含B细胞表位或肽半抗原的目标抗原蛋白结合的抗体，可以减低目标蛋白的含量和/或活性,显著调节Th1/Th2平衡失调，由Th1偏向Th2转移，显著改善疾病的发病症状。以及含有该刺激多肽及其衍生物和类似物和偶联蛋白和融合蛋白及其免疫产生的特异抗体和抗体衍生物和类似物的药物制剂，以及提供的该刺激多肽及含此刺激多肽的多肽免疫原用于重组微生物和转基因动物或植物及其细胞，使它们作为生产工厂，生产含此刺激多肽的多肽免疫原和融合蛋白和偶联蛋白或制作口服疫苗或生产特异抗体及其衍生物和类似物的药物制剂，在预防和治疗Th1占优势诱导的疾病风湿病，多发硬化症、糖尿病、肾病、动脉粥样硬化、高血脂、高尿酸、老年痴呆、眼病、足病、心血管等方面具有重要应用价值，产生的特异抗体及其衍生物和类似物或改造物可供防治相关疾病或检测相关目标抗原蛋白，开发为药物或检测试剂。
进一步，当B细胞表位或合成半抗原选择为人二肽基肽酶4(dipeptidyl peptidase 4，DPP4)的优势B细胞抗原表位或合成肽半抗原，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原D41-IP(D41-IA2(5)-P2-1),D41-2-IP和D41-3-IP或将其连接于IA2(5)与P2中间，形成IA2(5)-D41-P2-1等肽免疫原,在免疫正常小鼠后能产生高水平抗体，但是不引起低血糖，具有安全性；在免疫糖尿病模型小鼠后能产生特异性的与DPP4结合的抗体(属于Th2亚型)，升高Th2类细胞因子，调节Th1/Th2平衡，升高GLP-1量，升高胰岛素量，保护胰岛细胞，降低血糖，能为治疗与DPP4异常、血糖异常、血糖代谢紊乱及Th1/Th2失衡引起的糖尿病、肾病、动脉粥样硬化、老年痴呆、眼病、足病、心血管等疾病提供新途径；含DPP4优势B细胞抗原表位的多肽疫苗可以开发防治糖尿病，包括1型糖尿病，2型糖尿病，妊娠期糖尿病等类型糖尿病。也可以用于制备抗体及Th2抗体供治疗相关疾病或检测用。
进一步，当B细胞表位或合成半抗原选择为人黄嘌呤氧化还原酶(xanhtine oxidoreductase，XOD)的优势B细胞抗原表位或合成肽半抗原，将其与该刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原O-IA2(5)-P2,O-IA2(5)-P2-1，O2-IA2(5)-P2-2等多肽免疫原，或将其连接于IA2(5)与P2中间，形成IA2(5)-O-P2，在免疫小鼠后能产生特异性的与黄嘌呤氧化酶结合的抗体(属于Th2亚型)，升高Th2类细胞因子，调节Th1/Th2平衡，升高胰岛素量，保护胰岛细胞，降低血糖，降低黄嘌呤氧化酶活性，增显著增强抗氧化能力。可以开发治疗糖尿病并发症的疫苗和抗体。以多肽免疫原免疫动物，再通过杂交瘤技术制备的单克隆抗体属于Th2亚型，给药治疗高尿酸模型鼠，不降低动物血糖，可以特异性的与黄嘌呤氧化酶结合，升高Th2类细胞因子，调节Th1/Th2平衡，降低黄嘌呤氧化酶活性、降低血清尿酸和肌酐量，增加尿 酸和肌酐排泄，保护肾脏。可以开发用于制备抗体及Th2抗体供治疗相关疾病或检测用。
进一步，当B细胞表位或合成半抗原选择为人HMG-CoA还原酶(是肝细胞合成胆固醇过程中的限速酶)的优势B细胞抗原表位或合成肽半抗原，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原HIP1和H24IP1等肽免疫原，在免疫小鼠后能产生特异性的与HMG-CoA还原酶结合的抗体(属于Th2亚型)，降低HMG-CoA还原酶酶活,降低血脂，降低尿酸，升高SOD等等，可以开发用于制备抗体及Th2抗体供治疗疾病或检测用。
进一步，当B细胞表位或肽半抗原选择为人尿酸转运蛋白1(URAT1)的优势B细胞抗原表位，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原UIP-1，在免疫小鼠后能产生特异性的与URAT1结合的抗体(属于Th2亚型)，降血糖，增加尿酸排泄，降低血清尿酸和肌酐，保护肾脏。可以开发用于制备抗体及Th2抗体供治疗相关疾病或检测用，具有重要理论价值和广阔应用前景，有重要社会和经济效益。
当选择人Th1占优势的Th1/Th2失衡相关疾病的目标抗原蛋白如类风湿性关节炎，老年痴呆等的优势B细胞抗原表位或合成肽半抗原，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原，在免疫小鼠后能产生特异性的与含B细胞抗原表位的关键抗原蛋白结合的抗体(属于Th2亚型占优势)，改善疾病症状。可以开发用于制备抗体及Th2抗体供治疗相关疾病或检测用。当选择其他相关疾病的目标抗原蛋白，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原，在免疫小鼠后能产生特异性的与含B细胞抗原表位的关键抗原蛋白结合的抗体，可供治疗和开发检测试剂。
当选择其他的目标抗原蛋白的优势B细胞抗原表位或合成肽半抗原，将 其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原，在免疫小鼠后能产生特异性的与含B细胞抗原表位的关键抗原蛋白结合的抗体(属于Th2亚型占优势)，可以开发用于制备多种抗体及Th2抗体供检测用。具有重要理论价值和广阔应用前景，有重要社会和经济效益。
附图说明
为了使本发明目的，技术方案和优点清楚，结合附图对本发明进行进一步详细描述，其中：
图1为本发明含该刺激多肽的多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1)(简写B-IP)，B-IA2(5)-P2-2(简写B-2-IP)，IA2(5)-B-P2-1等(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、D41-2-IP、O-IA2(5)-P2、O-IA2(5)-P2-1、IA2(5)-O-P2、H24IP1、HIP1和UIP-1)免疫C57BL/6J雄鼠后血清产生的特异性抗体的ELISA和效价检测结果；
图2为本发明含该刺激多肽的多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1(简写B-IP)，B-IA2(5)-P2-2简写B-2-IP)，IA2(5)-B-P2-1等(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1、HIP1和UIP-1)免疫C57BL/6J雄鼠后血清产生的特异性抗体亚型的ELISA检测结果；
图3为含该刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1)免疫C57BL/6J雄鼠后的细胞因子检测结果；
图4为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶)的优势B细胞表位或肽半抗原时分别构成多肽免疫原 D41-IP、O-IA2(5)-P2-1)免疫C57BL/6J雄鼠后血清产生的特异性抗体分别与人DPP4(图4A，Lane 1:marker；Lane 2:DPP4；Lane 3:D41-BSA；Lane 4:BSA；Lane 5:L-ASP无关蛋白质)和人黄嘌呤氧化酶(图4B，1:marker；Lane 2:BSA；Lane 3:人黄嘌呤氧化酶)和牛黄嘌呤氧化酶片段(图4C，1:marker；Lane 2:牛黄嘌呤氧化酶片段；Lane 3:O-BSA；Lane 4:BSA；Lane5:L-ASP无关蛋白质)特异结合的Western Blot实验结果图；
图5为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、D41-2-IP、O-IA2(5)-P2、O-IA2(5)-P2-1、IA2(5)-O-P2、HIP1和H24IP1)免疫C57BL/6J雄鼠后分别抑制人DPP4(图5A)、嘌呤氧化酶(图5B)及HMG-CoA还原酶酶活性(图5C)的结果图；
图6为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1，B-IA2(5)-P2-2，IA2(5)-B-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1和UIP-1(图6A)，HIP1和H24IP1(图6B)免疫C57BL/6J雄鼠后降血糖结果图；
图7为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1免疫C57BL/6J雄鼠后血清ELISA检测GLP-1结果；
图8为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为DPP4、黄嘌呤氧化酶、HMG-CoA还原酶的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1、UIP-1、HIP1、H24IP1)免疫C57BL/6J雄鼠后血脂，尿酸和肌酐的检测结果；
图9为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为黄嘌呤氧化酶、HMG-CoA还原酶的优势B细胞表位或肽半抗原时分别构成多肽免疫原O-IA2(5)-P2-1、UIP-1、HIP1、H24IP1)免疫C57BL/6J雄鼠后抗氧化应激的各项指标GSH-PX、CAT、SOD、MDA的检测结果；
图10为本发明含刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1)的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP(图10C)、O-IA2(5)-P2-1(图10D)、HIP1(图10E)和UIP-1(图10F)免疫C57BL/6J雄鼠后胰腺免疫组化的检测结果；图10A为溶剂，图10B为IA2(5)-P2-1组；
具体实施方式
实施例1：一种含T辅助细胞表位(Th表位)的多肽IA2(5)-P2、IA2(5)-P2-1(简写IP)、IA2(5)-P2-2(简写2-IP)和IA2(5)-B-P2-1等的制备和合成。
选用IA-2的一个线形B细胞表位IA2(5)肽段，氨基酸序列1见SEQ ID NO.1，P277的C端的Th2表位P2肽段，氨基酸序列2见SEQ ID NO.2，运用分析软件及多次大量STZ糖尿病模型鼠实验的筛选分析鉴定修改，确定将两个表位直接或以柔性肽相连，将IA2(5)肽段FEYQD的C端和Th2表位P2肽段的N端直接或通过柔性肽或连接肽段的连接形成IA2(5)-P2、IA2(5)-P2-1(简写IP)、IA2(5)-P2-2(简写2-IP)等，柔性肽或连接肽氨基酸序列分别见SEQ ID NO.3和SEQ ID NO.4。IA2(5)-P2、IA2(5)-P2-1IA2(5)-P2-2氨基酸序列分别见SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7；或将IA2(5)肽段FEYQD的C端和B细胞表位或肽半抗原通过柔性肽或连接肽共价连接后，再和Th2表位P2肽段的N端通过柔性肽或连接肽段的连接形成多肽免疫原IA2(5)-B-P2，IA2(5)-B-P2-1等，IA2(5)-D41-P2，IA2(5)-D41-P2-1和IA2(5)-O-P2的氨基酸序列分 别见SEQ ID NO.8，SEQ ID NO.9和SEQ ID NO.10。肽采用FMOC固相合成法合成，HPLC检测纯度＞90％。
实施例2：B细胞表位或合成肽半抗原B的制备和合成。
运用B细胞抗原表位分析软件以及在线分析工具，以单参数为基础，结合二级结构预测及空间结构特点，分别从一个目标抗原蛋白质(如：DPP4或XOD或HMG-CoA还原酶和尿酸转运蛋白1)的100多个表位中选出多个优势B细胞抗原表位或肽半抗原B，通过与IA2(5)-P2，IA2(5)-P2-1(简写IP)、IA2(5)-B-P2-1、等以柔性肽连接，经过免疫动物实验筛选任何再改造再筛选的过程，获得免疫原性高的B细胞抗原表位或合成肽半抗原B。肽均采用FMOC固相合成法合成，HPLC检测纯度＞90％。
实施例2-1当B为人二肽基肽酶4(dipeptidyl peptidase 4，DPP4)的优势B细胞抗原表位,制备方法是从其100多个表位中选出D41，D42，D43，D44-----等等多个优势B细胞抗原表位或肽半抗原B，通过与IA2(5)-P2，IA2(5)-P2-1(简写IP)、IA2(5)-P2-2(简写2-IP)、IA2(5)-B-P2等的N端以柔性肽连接形成肽免疫原，经过免疫动物实验筛选获得免疫原性高的B细胞抗原表位或肽半抗原D41，D42，D43等等。D41是DPP4的一段特异性多肽88VFLENSTFDE97,共10个氨基酸残基，氨基酸序列见SEQ ID NO.11。
实施例2-2当B为人黄嘌呤氧化还原酶(xanhtine oxidoreductase,XOD)的优势B细胞抗原表位，从其100多个表位中选出O1，O2，O3-----等等多个优势B细胞抗原表位或肽半抗原B，通过与IA2(5)-P2，IA2(5)-P2-1(简写IP)、IA2(5)-P2-2(简写2-IP)、IA2(5)-B-P2的N端以柔性肽连接形成肽免疫原，经过免疫动物实验筛选任何再改造再筛选的过程，获得免疫原性高的B细胞抗原表位或肽半抗原O，O1等等。O是黄嘌呤氧化还原酶的一段特异性多肽 1101RLEPYKKKNPS1111,共11个氨基酸残基，氨基酸序列分别见SEQ ID NO.12。
实施例2-3当B为人HMG-CoA还原酶(是肝细胞合成胆固醇过程中的限速酶，从其100多个表位中选出H1，H2，H3-----等等多个优势B细胞抗原表位或肽半抗原B，通过与IA2(5)-P2，IA2(5)-P2-1、IA2(5)-P2-2的N端以柔性肽连接形成肽免疫原，经过免疫动物实验筛选获得免疫原性高的B细胞抗原表位或肽半抗原H，H1等。H和H1是黄嘌呤氧化还原酶的一段特异性多肽,氨基酸序列见SEQ ID NO.13和SEQ ID NO.14。
实施例2-4当B为人尿酸转运蛋白1(URAT1)的优势B细胞抗原表位，从其100多个表位中选出U1，U2，U3-----等等多个优势B细胞抗原表位或肽半抗原X，通过与IA2(5)-P2，IA2(5)-P2-1、IA2(5)-P2-2等等以柔性肽连接，经过免疫动物实验筛选获得免疫原性高的B细胞抗原表位或肽半抗原U1，U2，其采用FMOC固相合成法合成，HPLC检测纯度＞90％。
实施例2-5当B为Th1占优势的Th1/Th2失衡相关疾病的关键抗原蛋白的优势B细胞抗原表位，从其100多个表位中选出B1，B2，B3-----等等多个优势B细胞抗原表位或合成肽半抗原B，通过与刺激肽IA2(5)-P2，IA2(5)-P2-1、IA2(5)-P2-2等以柔性肽连接形成肽免疫原，经过免疫动物实验筛选获得免疫原性高的B细胞抗原表位或肽半抗原B1，B2，其采用FMOC固相合成法合成，HPLC检测纯度＞90％。
实施例3：多肽免疫原B-IA2(5)-P2、B-IA2(5)-P2-1(简写B-IP)、B-IA2(5)-P2-2(简写B-2-IP)(B为B细胞表位或合成肽半抗原)的制备和合成。
运用B细胞抗原表位分析软件以及在线分析工具，以多个单参数为基础，结合二级结构预测及空间结构特点，从一个目标抗原蛋白质(如：DPP4或 XOD或HMG-CoA还原酶和尿酸转运蛋白1等等)的100多个表位中选出多个优势B细胞抗原表位或肽半抗原B，将B的C端和IA2(5)-P2-1的N端连接，构成B-IA2(5)-P2、B-IA2(5)-P2-1、B-IA2(5)-P2-2、IA2(5)-B-P2，IA2(5)-B-P2-1等多肽免疫原，经过软件分析和动物实验多次筛选和改造，获得免疫原性高的含发明的刺激多肽IA2(5)-P2、IA2(5)-P2-1(简写IP)、IA2(5)-P2-2(简写2-IP)的多肽免疫原B-IA2(5)-P2、B-IA2(5)-P2-1、B-IA2(5)-P2-2、IA2(5)-B-P2，IA2(5)-B-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1)的优势B细胞表位或合成肽半抗原时分别构成多肽疫苗D41-IP(氨基酸序列见SEQ ID NO.15)、D41-2-IP(氨基酸序列见SEQ ID NO.16)、O-IA2(5)-P2(氨基酸序列见SEQ ID NO.17)、O-IA2(5)-P2-1(氨基酸序列见SEQ ID NO.18)、IA2(5)-O-P2-1、HIP1(氨基酸序列见SEQ ID NO.19)、H24IP1(氨基酸序列见SEQ ID NO.20)和UIP-1。
上述叙述的多肽均采用FMOC固相合成法合成，HPLC检测纯度＞90％。分析用肽采用FMOC固相合成法合成，HPLC检测纯度＞95％，再和蛋白质BSA或KLH偶联。
实施例中的实验方法，如无特别说明，均为常规方法。所用试剂盒均按照制造产品厂商所建议的条件进行。
实施例4：含本发明刺激多肽的多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1、B-IA2(5)-P2-2、IA2(5)-B-P2，IA2(5)-B-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1的优势B细胞表位或肽半抗原)D41-IP、D41-2-IP、O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1、H24IP、HIP1和UIP-1)的免疫原性和药效学评价。
实施例4-1、含本发明刺激多肽的多肽免疫原B-IA2(5)-P2， B-IA2(5)-P2-1、B-IA2(5)-P2-2、IA2(5)-B-P2，IA2(5)-B-P2-1(B分别为人DPP4、黄嘌呤氧化酶、HMG-CoA还原酶和尿酸转运蛋白1的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、D41-2-IP、O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1、H24IP、HIP1和UIP-1)免疫STZ诱导的糖尿病模型鼠的免疫原性和药效学评价。
实施例4-1-1.STZ诱导的糖尿病模型鼠的的建立
采用小剂量连续多次腹腔注射链脲佐菌素(STZ)诱导小鼠糖尿病(DM)模型。4周龄雄性C57BL/6J小鼠(购自扬州大学比较医学中心，许可证号：SCXR(苏)2012-0004。)用0.1M pH4.4柠檬酸盐缓冲液配置STZ浓度为7.5mg/ml，按50mg/kg剂量腹腔注射，每日1次，连续注射5次，于造模后第3、7、14天测定血糖值，以空腹血糖连续两次≥11.1mmol/L为成模标准。
实施例4-1-2.造模成功后动物免疫的治疗实验
100多只糖尿病模型小鼠随机分成13组，每组10只，分别为：placebo组(溶剂对照组，阴性对照组)、IA2(5)-P2、IA2(5)-P2-1、IA2(5)-P2-2对照组、D41-IP、D41-2-IP、O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1、H24IP、HIP1和UIP-1给药组。给药组药物浓度均为1mg/mL，给药量为100μg/只/次，即100μL药液；阴性对照组：100μl油剂/只/次。药物配置方法按1mg+40mg甘露醇溶于1ml20％Lipofundin的比例临时配置油剂。在模型成功(以血糖≥11.1mmol/L为准)后第2周开始进行动物免疫。每周给药1次，连续5次，再分别于7、9、11周免疫三次，共计8次。
实施例4-1-3.含本发明刺激多肽的多肽免疫原D41-IP、D4-2-IP、IA2(5)-D41-P2-1、IA2(5)-D41-P2、O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1、H24IP、HIP1和UIP-1分别免疫C57BL/6J雄鼠后血清产生的特异性抗体的ELISA和抗 体效价检测：
包被稀释液(0.05M碳酸钠-碳酸氢钠缓冲液，pH9.6)稀释X-BSA，以BSA作为对照组，分别配成终浓度为0.5mg/mL的溶液，96孔酶标板中每孔加入0.1mL,4℃包被过夜；弃去包被液，每孔加入5％脱脂奶粉(pH7.4PBS配制)封闭液，4℃满孔封闭过夜；弃去封闭液，每孔加入1:100,5％脱脂奶粉稀释的抗原肽免疫的小鼠血清0.1mL，37℃孵育1h；弃去血清，每孔用PBST(含0.5％吐温-20的PBS溶液)和蒸馏水间隔洗涤，每次3min，共洗涤6次；洗涤后，每孔加入5％脱脂奶粉稀释的辣根过氧化酶标记的羊抗小鼠IgG二抗0.1mL，稀释倍数1:5000，(效价测定时分别稀释1:100,1：500，1:1000,1:4000，1：6000，1：8000，1:10000，1：12000，1:14000，1：16000，1：18000，1:1,,20000，)37℃孵育1h；弃去二抗液，每孔用PBST和蒸馏水间隔洗涤，每次3min，共洗涤6次；洗涤后，每孔加入0.1mL TMB显色液，37℃反应30min后，加入2mol/L的H2SO4 0.1mL终止反应；双蒸水调零，酶标仪450nm波长下测定各孔的OD值。结果如图1所示，实验结果表明，含本发明多肽的多肽免疫原D41-IP、D41-2-IP、IA2(5)-O-P2、O-IA2(5)-P2-1、H24IP、HIP1和UIP-1分别免疫糖尿病小鼠后血清中产生针对B的特异性抗体量显著增加，与溶剂组和IA2(5)-P2-1、IA2(5)-P2-2组相比具有统计学差异(P＜0.001\P＜0.01\P＜0.05)。D41-IP、D41-2-IP、IA2(5)-D41-P2-1、O-IA2(5)-P2-1、HIP1、H24IP1和UIP-1的血清抗体效价均在8000到20000之间(见表1)，可以用于制备抗体，开发治疗药物和检测试剂。
表1 含刺激肽的肽免疫原免疫小鼠血清效价检测结果
Figure PCTCN2017114349-appb-000001
Figure PCTCN2017114349-appb-000002
实施例4-1-4.为本发明含刺激多肽的多肽免疫原D41-IP、O-IA2(5)-P2-1、HIP1和UIP-1分别免疫C57BL/6J雄鼠后产生的特异性抗体亚型的ELISA检测：
购自酶免生物，按照试剂盒说明书操作，每孔加入50ul标准品，或者5倍稀释的血清。每孔加入100uL酶标二抗，保鲜膜缠绕后37℃孵育60min。wash solution满孔洗板5次。每孔加入50uL底物A和50uL底物B，轻轻混匀，37℃避光孵育15min。每孔加入50uL终止液(2M H2SO4)(颜色由蓝变黄)。15min内在450nm波长处测定OD值。以标准品的OD值为横坐标，浓度值为纵坐标，绘制标准曲线，计算样品浓度。结果如图2所示，实验结果表明，B-IA2(5)-P2-1(包括D41-IP、O-IA2(5)-P2-1、HIP1和UIP-1)分别免疫糖尿病小鼠后血清中针对B的特异性Th2抗体IgG1和IgG2b量显著增加，D41-IP与溶剂组和IA2(5)-P2组相比IgG1和IgG2b量显著增加，具有统计学差异(P＜0.01)。O-IA2(5)-P2-1和UIP-1与IA2(5)-P2组相比IgG1和IgG2b量显著增加(P＜0.05)。可以用于制备Th2抗体(B表位与BSA或KLH偶联还可以制备Th1抗体)，可治疗Th1偏高疾病和检测特异抗原。
实施例4-1-5.为含本发明刺激多肽的的多肽免疫原D41-IP和O-IA2(5)-P2-1在免疫治疗STZ诱导的糖尿病C57BL/6J雄鼠模型后细胞因子检 测：
细胞因子测定根据试剂盒说明书进行。简要实验步骤为1、标准品和样品的稀释与加样。在96孔板上准确加入标准品50μL，或者5倍稀释的血清。2、温育，用封板膜封板后置于37℃温育30分钟。3、洗涤，弃去液体后甩干，每孔加满洗涤液，静置30s后弃去，如此重复5次，拍干。4、每孔加入酶标试剂50μl，空白孔除外。5、温育，操作同2。6、洗涤，操作同3。7、显色，每孔加入50μl底物A和50μl底物B，轻轻混匀，37℃避光孵育15min。8、终止，每孔加入50μl终止液(2M H2SO4)(颜色由蓝变黄)。9、测定，空白孔调零，450nm波长测量各孔的吸光度(OD值)。实验结果见图3，结果表明，D41-IP、O-IA2(5)-P2-1免疫C57BL/6J小鼠后，IL-10(图3)水平显著升高，IFN-γ(图3)水平显著降低，与溶剂组相比具有统计学意义(P＜0.01)说明Th2介导的免疫应答升高，Th1介导的免疫应答下降。这证明D41-IP、O-IA2(5)-P2-1可以调节糖尿病小鼠体内Th1/Th2失衡，更多的干预免疫平衡向Th2转化。D41-IP、O-IA2(5)-P2-1的免疫可用于治疗由体内Th1/Th2失衡引起的疾病，如1型糖尿病及其并发症，炎症反应等其他Th1/Th2失衡相关的疾病。
实施例4-1-5.含刺激多肽的多肽疫苗D41-IP、O-IA2(5)-P2-1分别免疫C57BL/6J雄鼠后血清产生的特异性抗体分别与人DPP4和黄嘌呤氧化酶特异结合的Western Blot实验：
依据Western Blot标准操作规程，将DPP4转移到PVDF膜上，转移条件为100V,5h；转移后的PVDF膜至于25mL封闭缓冲液中室温下摇动1h。15mL TBST洗3次(5min/次)；然后将PVDF膜浸泡于1:10稀释的抗原肽免疫小鼠血清中，室温孵育1-2h或4℃过夜，缓慢摇动，15mL TBST洗3次(5min/次)。再加入1:5000稀释的辣根过氧化物酶标记的羊抗鼠IgG，37℃孵育1h。PBST 洗膜3次，每次10min。加入DAB显色液进行显色。充分显色后将膜转移至去离子水中终止显色反应。结果如图4所示，结果表明：B-IA2(5)-P2-1(包括D41-IP、O-IA2(5)-P2-1)分别免疫小鼠后产生的血清中抗体能与含B的目标蛋白质抗原DPP4或黄嘌呤氧化酶及主要片段特异结合，而不和无关蛋白质结合，抗体可以开发供检测目标抗原及其片段用。
实施例4-1-6.含刺激多肽的多肽免疫原D41-IP、O-IA2(5)-P2-1、HIP1和H24IP1分别免疫C57BL/6J雄鼠后的血清抑制体外DPP4和黄嘌呤氧化酶活性及血清HMG-CoA还原酶测定：
实施例4-1-6-1.多肽免疫原免疫C57BL/6J雄鼠后的血清抑制体外酶活性的抑制率测定按照江南大学苗雷文献方法测定，总反应体系100ul,将血清，缓冲液，酶于37摄氏度下分别孵育30分钟；设4组,按照酶，缓冲液，底物的顺序加入96孔板，37℃恒温培养箱反应60min，测定405nm处的吸光度。比较各组OD405的值，计算抑制率。结果如图5A所示，结果表明：与溶剂和IA-2(5)-P2-1比较，D41-IP免疫小鼠后产生的血清能显著抑制体外DPP4的活性(P＜0.001)。
实施例4-1-6-2.HIP1免疫C57BL/6J雄鼠后的血清HMG-CoA还原酶活性测定试剂盒购自上海宝曼公司，测定方法按照说明书进行。结果如图5C所示，结果表明：与溶剂和IA-2(5)-P2-1比较，HIP1免疫小鼠后产生的血清中HMG-CoA还原酶活性显著降低(P＜0.01)，H24IP1免疫小鼠后产生的血清中HMG-CoA还原酶活性显著降低(P＜0.05).
4-1-6-3.O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1免疫C57BL/6J雄鼠后的血清抑制体外黄嘌呤氧化酶活性的抑制率测定按照文献方法测定，总反应体系2.45mL。血清取10uL加40uL缓冲液稀释到50uL。不加抑制剂的两管(OD2，OD4)按表格试剂加完之后，立即测量吸光度。加抑制剂的两管(OD1， OD3)，先加缓冲液，再加抑制剂，再加酶，37℃孵育20分钟后，加入底物后混匀，60秒后测定294nm处的吸光度。比较各组OD294的值，计算抑制率。结果如图5B所示，结果表明：与溶剂比较，O-IA2(5)-P2、IA2(5)-O-P2、O-IA2(5)-P2-1免疫小鼠后产生的血清能小显著抑制体外黄嘌呤氧化酶的活性(P＜0.01)
Figure PCTCN2017114349-appb-000003
实施例4-1-7-1.含刺激肽的多肽免疫原D41-IP、D41-2-IP、IA2(5)-D41-P2-1、O-IA-2(5)-P2-1、H24IP、HIP1和UIP-1分别免疫C57BL/6J雄鼠后降血糖实验：
在模型成功(以血糖≥11.1mmol/L为准)后每周给药1次，连续5次，再分别于7、9、11周免疫三次，共计8次。每次免疫前将糖尿病小鼠断尾取血，取血时用血糖监测仪检测小鼠血糖水平。结果如图6所示，实验结果表明，B-IA2(5)-P2-1(包括D41-IP,D41-2-IP，O-IA2(5)-P2-1、HIP1和UIP-1)免疫糖尿病小鼠后可显著性降低小鼠血糖，D41-IP与溶剂组和IA-2(5)-P2组相比具有统计学差异(P＜0.001)。D41-2-IP，O-IA2(5)-P2-1、H24IP、HIP1和UIP-1与溶剂组相比具有统计学差异(P＜0.01)。可以开发用于治疗糖尿病。
实施例4-1-7-2.为含刺激肽的肽免疫原D41-IP在免疫防治NOD小鼠1型糖尿病模型后降血糖和死亡率、发病率检测：
30只雌性NOD小鼠(购自北京华阜康公司，许可证号： SCXK(京)2014-0004。)随机分3组，每组10只：溶剂组(阴性对照组)、P277组(阳性对照组)、D41-IP组。按照7,19,20,21,22,23,25,27,29给药。皮下多点免疫，四肢腋下和背部进行免疫。给药组药物浓度为1mg/mL，给药剂量100μg/只/次，阴性对照组：100mL油剂力保肪宁/只/次。肽D41-IP免疫治疗小鼠后，采用断尾取血的方法用血糖仪定期测定小鼠的血糖。经NOD雌性小鼠给予含有载体肽肽免疫原后，小鼠的血糖变化、发病率和死亡率情况如表2所示。结果显示：肽免疫原D41-IP和对照组比较，能显著控制NOD小鼠血糖，发病率和死亡率，最后小鼠血糖基本恢复正常，无发病率和死亡率。
表2肽免疫原免疫防治NOD小鼠的血糖、发病率和死亡率检测结果
Figure PCTCN2017114349-appb-000004
实施例4-1-8.含刺激多肽的多肽免疫原D41-IP、O-IA2(5)-P2-1分别免疫C57BL/6J雄鼠后血清ELISA检测GLP-1或胰岛素水平：
小鼠血清中GLP-1水平的测定按照试剂盒说明进行(酶免生物科技有限公司)。按照试剂盒说明在450nm波长处测定OD值。D41-IP、O-IA2(5)-P2-1分别免疫C57BL/6J雄鼠7次后，测定血清中GLP-1水平。实验结果如图7所示。结果表明，D41-IP免疫后，小鼠体内GLP-1水平显著升高，和溶剂组和IA2(5)-P2-1相比具有统计学差异(P＜0.01)。O-IA2(5)-P2-1免疫后，小鼠体内胰岛素水平显著升高，和溶剂组相比具有统计学差异(P＜0.05)。可以开发治疗 糖尿病。
实施例4-1-9.含刺激多肽的多肽免疫原O-IA2(5)-P2-1和HIP1和UIP-1免疫C57BL/6J雄鼠后血脂、尿酸、肌酐和抗氧化应激指标的检测；
小鼠血清血脂，尿酸和抗氧化应激的检测试剂由南京建成公司提供，按照试剂盒说明进行检测和计算。实验结果如图8和图9所示，实验结果表明，O-IA2(5)-P2-1和HIP1和UIP-1免疫小鼠后，小鼠血清尿酸和肌酐显著降低，抗氧化作用显著增强,具有统计学差异(P＜0.05)。HIP1免疫小鼠后，小鼠血清血脂显著降低,具有统计学差异(P＜0.05)。可以缓解糖尿病并发症。
实施例4-1-10.含刺激多肽的多肽免疫原D41-IP,O-IA2(5)-P2-1、HIP1和UIP-1免疫治疗C57BL/6J雄鼠后胰腺的胰岛素免疫组化鉴定；
动物实验结束时，取实验组和对照组动物组织尽可能新鲜，PBS(磷酸盐缓冲液)洗，取小于0.5cm×0.5cm×0.1cm组织块固定和包埋，切片，切片上滴加胰岛素抗体处理，滴加生物素化第二抗体(IgG)处理，再滴加辣根酶标记链霉卵白素工作液(S-A/HRP)，37℃20分钟，0.1MPBS洗3次×5分钟；，DAB显色，洗涤，中性树脂封片，显微镜观察：选择实验组和对照组的阳性和阴性组织进行200×的显微照相。实验结果如图10所示，实验结果表明，D41-IP，O-IA2(5)-P2-1和HIP1和UIP-1免疫小鼠后，与溶剂和IA2(5)-P2-1(IP)对照相比，小鼠胰腺得到显著改善，胰岛分泌胰岛素量明显增加。可以开发保护胰腺药物。
实施例4-2、含本发明刺激多肽的多肽免疫原B-IA2(5)-P2-1(B分别为人DPP4、黄嘌呤氧化酶的优势B细胞表位或肽半抗原时分别构成多肽免疫原D41-IP、O-IA2(5)-P2-1免疫1型糖尿病NOD模型鼠的免疫原性和药效学评价。
按照STZ模型鼠同样给药方法，结果显示，D41-IP多肽免疫原能有效 控制糖尿病发病，作用和机理同STZ模型鼠。
结论：本发明的含T辅助细胞表位(Th表位)的刺激多肽的N端和B细胞表位或肽半抗原---一种目标抗原蛋白的目标抗原位点B的C端共价连接，形成为多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1，B-IA2(5)-P2-2，IA2(5)-B-P2-1等可以显著改善目标抗原位点(B细胞表位或肽半抗原)的免疫原性并用于制备特异的与含B细胞表位或肽免疫原的目标抗原蛋白结合的抗体及Th2亚型抗体，含有该刺激多肽的多肽免疫原和基于其生产的抗体及Th2亚型特异抗体以及含有该多肽及其衍生物和类似物及其产生的特异抗体和抗体衍生物和类似物的药物制剂，在预防和治疗Th1占优势诱导的疾病等方面具有重要应用价值，抗体及Th2亚型特异抗体及其衍生物和类似物可检测相关目标抗原蛋白，开发为检测试剂。当B为DPP4的优势B细胞表位时，产生特异的高效价的能和DPP4结合的抗体及Th2亚型抗体，抑制DPP4活性，升高GLP-1、保护β细胞，增加胰岛素分泌，显著降血糖，降低尿酸和肌酐，可用于DPP4异常、血糖异常、血糖代谢紊乱及Th1/Th2失衡引起的糖尿病、肾病、动脉粥样硬化、老年痴呆、眼病、足病、心血管等疾病治疗上的应用。此外，制备的抗体及Th2亚型抗体可开发供治疗和检测用。当B为人黄嘌呤氧化还原酶(xanhtine oxidoreductase,XOR)的优势B细胞抗原表位，将其C端与IA2(5)-P2-1相连接的多肽免疫原在免疫小鼠后能产生特异性的与黄嘌呤氧化酶结合的抗体(属于Th2亚型占优势抗体)，升高Th2类细胞因子，调节Th1/Th2平衡，升高胰岛素量，保护胰岛细胞，降低血糖，降低黄嘌呤氧化酶活性，增显著增强抗氧化能力。可以开发治疗糖尿病并发症的疫苗和抗体。以此类含刺激多肽的免疫原免疫动物，可以通过杂交瘤技术、抗体库技术、转基因动物技术、单细胞技术等制备单克隆抗体及基因工程抗体，其属于Th2亚型，给药治疗高尿酸模型鼠， 不降低动物血糖，可以特异性的与黄嘌呤氧化酶结合，升高Th2类细胞因子，调节Th1/Th2平衡，抑制黄嘌呤氧化酶活性、增加胰岛素分泌，降低血清尿酸和肌酐量，增强抗氧化作用，保护肾脏。可以开发用于制备抗体及Th2抗体供治疗疾病或检测用。当B为人HMG-CoA还原酶(是肝细胞合成胆固醇过程中的限速酶，抑制HMG-CoA还原酶能阻碍内源性胆固醇合成)，的优势B细胞抗原表位或肽半抗原，将其C端与IA2(5)-P2-1相连接的多肽免疫原HIP1和H24IP1在免疫小鼠后能产生特异性的与HMG-CoA还原酶结合的抗体(属于Th2亚型占优势抗体)，降低HMG-CoA还原酶酶活,降低血脂，增加胰岛素分泌，降低血糖，增强抗氧化作用，可以开发用于制备抗体及Th2抗体供治疗疾病或检测用。当B为人尿酸转运蛋白1(URAT1)，的优势B细胞抗原表位，将其C端与IA2(5)-P2-1相连接的多肽免疫原UIP-1在免疫小鼠后能产生特异性的与URAT1结合的抗体(属于Th2亚型占优势抗体)，增加胰岛素分泌降低血清尿酸和肌酐，保护肾脏。可以开发用于制备抗体及Th2抗体供治疗相关疾病或检测用。当选择人Th1占优势的Th1/Th2失衡相关疾病的关键抗原蛋白如类风湿性关节炎，老年痴呆的优势B细胞抗原表位，将其与刺激多肽通过柔性肽或连接肽相连接形成多肽免疫原，在免疫小鼠后能产生特异性的与含B细胞抗原表位的关键抗原蛋白结合的抗体(属于Th2亚型占优势抗体)，改善疾病症状。可以开发用于制备抗体及Th2抗体供治疗相关疾病或检测用。当选择其他Th2占优势的Th1/Th2失衡相关疾病的目标抗原蛋白，将其与刺激多肽的N端通过柔性肽或连接肽相连接形成多肽免疫原，在免疫小鼠后能产生特异性的与含B细胞抗原表位的关键抗原蛋白结合的抗体，可供开发检测试剂，具有重要理论价值和广阔应用前景，有重要社会和经济效益。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽 管通过参照本发明的某些优选实施例已经对本发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。

Claims (10)

  1. 一种多肽，其特征在于该多肽含有一个多肽序列，该多肽序列含有氨基酸序列1和氨基酸序列2，氨基酸序列1和氨基酸序列2通过直接或柔性肽或连接肽段的连接形成该种多肽，所述氨基酸序列1是胰岛素瘤相关蛋白IA-2的B细胞表位IA2(5),所示氨基酸序列组成见SEQ ID No.1,所述氨基酸序列2是P277的C端的Th2细胞表位P2，所示氨基酸序列组成见SEQ ID No.2。
  2. 根据权利要求1所述的多肽，当IA2(5)的C端和P2肽段的N端直接或通过柔性肽或连接肽段的连接形成一种多肽，IA2(5)-P2，IA2(5)-P2-1(简写IP)，IA2(5)-P2-2(简写2-IP)，所示氨基酸序列组成见SEQ ID No.5，SEQ ID No.6，SEQ ID No.7。
  3. 根据权利要求1所述的多肽，当IA2(5)的C端和目标抗原的B细胞表位或肽半抗原的N端通过Gly，GlyGly，GlyGlyGly或其他小于3个氨基酸残基的连接肽连接后，再和Th2表位P2肽段的N端通过柔性肽或连接肽段的连接形成多肽免疫原IA2(5)-B-P2，IA2(5)-B-P2-1(简写B-IP),IA2(5)-B-P2-2(简写B-2-IP)等,当其中B是二肽基肽酶4的B细胞表位D41(所示氨基酸序列组成见SEQ ID No.11)并以Gly连接IA2(5),再以柔性肽或连接肽段连接P2时，多肽免疫原所示氨基酸序列组成见SEQ ID No.8和SEQ ID No.9,当B是黄嘌呤氧化酶的B细胞表位O(所示氨基酸序列组成见SEQ ID No.12)时，多肽免疫原所示氨基酸序列组成见SEQ ID No.10。
  4. 根据权利要求1-3所述的柔性肽和连接肽接头为Xaa1Xaa2Xaa3------Xaan，Xaa1Xaa2Xaa3Xaa4Xaa5、Xaa1Xaa2Xaa3Xaa4、Xaa1Xaa2Xaa3、Xaa1Xaa2、Xaa、GGGGG、GGGG、GGG、GGS、GG、G、K、KK或其他短肽,Xaa1Xaa2Xaa3Xaa4Xaa5和Xaa1Xaa2Xaa3Xaa4所述氨基酸序列见SEQ ID No.3和SEQ ID No.4，Xaan为任一种氨基酸残基，n优选阿拉伯数字1-20。
  5. 根据权利要求1-2所述的多肽，目标抗原的B细胞表位或肽半抗原通过Gly、GlyGly、GlyGlyGly或其他小于3个氨基酸残基的连接肽和刺激多肽IA2(5)-P2，IA2(5)-P2-1，IA2(5)-P2-2共价连接，形成多肽免疫原B-IA2(5)-P2，B-IA2(5)-P2-1(简写B-IP)，B-IA2(5)-P2-2(简写B-2-IP)，当B是二肽基肽酶4的B细胞表位D41(氨基酸序列组成见SEQ ID No.11)并以Gly连接多肽时，形成的多肽免疫原所示氨基酸序列组成见SEQ ID No.15和SEQ ID No.16,当B是黄嘌呤氧化酶的B细胞表位O(氨基酸序列组成见SEQ ID No.12)并以Gly连接多肽时，形成的多肽免疫原所示氨基酸序列组成见SEQ ID No.17和SEQ ID No.18,当B是HMG-CoA还原酶的B细胞表位H和H1(氨基酸序列组成见SEQ ID No.13和SEQ ID No.14)并以Gly连接多肽时，形成的多肽免疫原所示氨基酸序列组成见SEQ ID No.19和SEQ ID No.20。
  6. 根据权利要求3和5所述的含B细胞表位或肽半抗原的目标抗原，包括但是不限于Th1偏高疾病的相关的目标抗原。
  7. 根据权利要求1-6所述的多肽和多肽免疫原及衍生物及其偶联蛋白和融合蛋白和串并联物也可以用于重组微生物和能转基因的动物或植物及其各种细胞，使它们作为生产工厂，能够生产含此B细胞表位及含有此表位的多肽免疫原和偶联蛋白抗原及其融合蛋白或制作口服疫苗或生产特异的抗体及Th2抗体及其衍生物和类似物及药物。
  8. 包含免疫学有效量的权利要求1-7中所述的含此多肽的多肽免疫原及其偶联蛋白和融合蛋白的组合物或串并联物，其特征在于：还包含药学上可接受的载体和药学上可接受的辅料。包含此多肽和多肽免疫原及其偶联蛋白和融合蛋白及由此抗原肽改构得到的抗原肽为有效成分的制剂，多肽及免疫原与药学上可以接受的载体连接的偶联蛋白或融合蛋白为有效成分的制剂的注射 剂、缓释剂、皮下埋植剂、片剂、粉剂、颗粒剂、胶囊或者口服液。
  9. 根据权利要求1-8所述的该多肽和多肽免疫原以及其改构得到的免疫原及其免疫原组合物串并联物等用作免疫刺激物即显著增强B细胞表位或肽半抗原的免疫原性，抑制目标抗原的活性或功能，此抗体是Th2亚型，其特征在于：可用于制备药物治疗Th1偏高相关疾病。用于制备能和目标抗原特异性结合的各种类型抗体抑制剂及基于其用各种方法制备和改造的抗体和衍生物及类似物。
  10. 根据权利要求1-9所述的含该多肽的免疫原和基于其生产的抗体及衍生物和改造物，可用于制备药物防治目标抗原异常、血糖和尿酸异常、细胞因子异常、氧化应激异常、血糖、血脂、尿酸、肌酐代谢紊乱及Th1偏高引起的疾病包括但不限于糖尿病及其并发症，肾病、高血脂、高尿酸血症、动脉粥样硬化、风湿性关节炎、老年痴呆、眼病、足病、心血管等相关疾病的预防和治疗，保护胰腺，及用于检测相关特异目标抗原或片段，开发检测试剂。
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