CN109836505A - One kind is used to connect the carrier peptides of B epitope or haptens and the immunogene containing carrier peptides in medicine and immunologic purposes - Google Patents

Abstract

The present invention provides the similar carrier peptides of a class formation, B cell epitope can be connected or peptide haptens constitutes peptide based immunogens, simplify it is immune prepared with antigen, have important use in medicine and immunology.Different mouse are immunized in peptide based immunogens containing carrier peptides of the present invention, significantly increase the immunogenicity of B cell epitope or peptide haptens, generate the Th2 antibody of high price with target antigen albumen specific bond, with this peptide based immunogens it is immune after special Th2 antibody can be prepared by hybridoma technology or antibody library and the antibody production techniques such as unicellular, significantly increase the immunological regulation for being biased to Th2, inhibit enzyme activity or GAP-associated protein GAP function, improves illness.Carrier peptides provided by the invention and the immunogene containing carrier peptides can be used for the animal or plant and its cell of recombinant microorganism and transgenosis simultaneously, make it as production plant, vaccine or production oral vaccine or production specific antibody and modification can be produced, for preventing and treating that Th1 is dominant or peptide epitopes related disease includes but is not limited to diabetes and complication etc. and for developing detection reagent.

Description

Immunogene of the one kind for connecting the carrier peptides of B epitope or haptens and containing carrier peptides In medicine and immunologic purposes

Technical field

The present invention relates to immunology, pharmacy and medicine related fieldss.The present invention relates to the carriers with a kind of similar structure The immunogene and its application of peptide and the peptide containing examples of such carriers, especially examples of such carriers peptide are used for A kind of target antigen site of target antigen albumen, which is covalently attached, becomes peptide based immunogens, and target antigen site can be enhanced, and (B is thin Born of the same parents' epitope or peptide haptens) immunogenicity and can be used for preparing prevent and treat property vaccine and specifically with epitope containing B cell Or or the protein bound multiple types antibody of target antigen of peptide based immunogens of peptide haptens (can be mostly anti-, monoclonal antibody And based on anti-with various genetic engineerings of such peptide based immunogens after immune with antibody library and the antibody production techniques preparation such as unicellular Body), peptide based immunogens and its coupling protein, fusion protein and polymer, conjugate containing the carrier peptides and based on such After peptide based immunogens are immune with the various genetic engineering specific antibodies of antibody library and the antibody production techniques preparation such as unicellular and Derivative and analogue containing the carrier peptides and each species specific antibodies generated on its basis and antibody derivatives and similar The drug and preparation of object are preventing and treating the sides such as a variety of diseases of CD4+ attenuating and the increase of CD8+T cell and Th1 dominance Face has significant application value, by each species specific antibodies and its derivative and similar of the peptide based immunogens preparation containing this carrier peptides Object can also be used in the main sections for detecting related objective antigen protein or target antigen albumen, can be developed into detection reagent.

Background technique

Human and animal equally exists two helper lymphocyte T subgroups, Th1 cell mainly generate Th1 class cell because Sub- IL-2, TNF-beta and IFN-γ；Th2 cell mainly generates IL-4, IL-5, IL-6, IL-10 and IL-13.Under normal circumstances, Helper lymphocyte T subgroup Th1/Th2 cell is in equilibrium state, but when body generation dysfunction, often shows to balance It is biased to a wherein side, this is " drift of Th1/Th2 balance ".Traditionally Th1 and its cell factor dominant state are called Th1 state, Th2 and its cell factor dominant state are known as Th2 state.Currently judge Th1 in disease immune pathogenesis Or Th2 cytological effect is that leading method can also be according to periphery mainly according to the expression of inflammatory tissue local cytokine The variation of Serum Antibody reaction subclass is judged.According to interference Th1 or Th2 cytological effect various links after Th1 or The variation of Th2 cytological effect is to judge to have 1 type glycosuria to the higher relevant human infection's disease of Th1 and from immunity disease Disease and complication and related disease, atherosclerosis, cardiovascular disease, senile dementia, kidney trouble, ephritis, rheumatoid joint Inflammation, adjuvant arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, erythema nodosum, habituation stream Produce etc..Lymphocyte includes T lymphocyte (CD3+), bone-marrow-derived lymphocyte (CD3-CD19+), NK cell (CD3-CD16+CD56 +), wherein T cell is the main composition of lymphocyte.CD3+ lymphocyte represents full T lymphocyte, it includes assisting/luring Lead T lymphocyte (CD3+CD4+), inhibition/Cytotoxic T lymphocytes (CD3+CD8+), apoptosis subgroup (CD95+) etc..Auxiliary/ Inducer T lymphocyte (CD3+CD4+) i.e. CD4+T lymphocyte is CD3 and CD4 all double positive cells, inhibition/cell toxicant T lymphocyte (CD3+CD8+) i.e. CD8+T lymphocyte is that CD3 and CD8 are positive cell.CD4+T Leukopenia is common In malignant tumour, genetic immunodeficiency disease, AIDS, using immunosuppressor etc..CD8+T cytosis sees 1 type glycosuria It is disease, systemic loupus erythematosus, rheumatoid arthritis, chronic active hepatitis, infectious mononucleosis, pernicious swollen Tumor and other virus infections etc..The higher induction morbidity such as CD8+, Th1 class T cell and cell factor, aggravates the state of an illness；CD4+,Th2 The increases such as class T cell and cell factor can then mitigate illness, or even prevent morbidity, the inflammation of this and CD4+/CD8+ and Th1/Th2 Property and anti-inflammatory response are related.Based on CD4+/CD8+⁺It is more and more with the relationship of Th1/Th2 drifting state and various diseases Research just tend to discovery, exploitation can reverse or stablize CD4⁺/CD8⁺With the drug and method of Th1/Th2 state.

Epitope is the basis of proteantigen, and proteantigen can embody its immunological characteristic by epitope, with regard to a certain egg For white matter, it, which contains, is also known as antigenic determinant (antigenic determinant, AD), T there are many epitope such as B cell epitope Auxiliary cell (Th) epitope etc., there are also inhibition epitope, toxicity epitope, cross reactivity epitopes etc..Select suitable table What position and it is connect by suitable method and other peptides or protein etc. and with intramolecular and molecule adjuvant and agent outside Type, how to be immunized etc. can just obtain ideal polypeptide or protein vaccine it is immune after just generate special high-titer (or high drop Degree) antibody has huge challenge.Th1 can generate IgG2a subclass antibodies in Mice Body, Th2 type can then generate IgG1 and IgG2b subclass antibodies, Th1 can generate IgG1 subclass antibodies in human body, and Th2 type can then generate IgG2a subclass antibodies, and Th2 is sub- Type antibody can increase the generation of Th2 cytokines and can treat the higher disease of Th1, alleviate illness, antibody and Th2 hypotype It can be used for detecting feature target antigen with Th1 subclass antibodies.

The determination of B cell epitope or peptide haptens is to the synthesis of polypeptide vaccine, the preparation of diagnostic reagent, monoclonal antibody The research work such as screening be of great significance, but B cell epitope or peptide haptens promote body to generate high titre antibody On condition that have strongly immunogenic, potency reaches the key of 8000 monoclonal antibodies made above or genetic engineering antibody.It is logical Often in order to increase the immunogenicity of B cell epitope or peptide haptens, by by B cell epitope or peptide hapten conjugation to KLH, The albumen such as BSA are building up to increase Th response on the fusion protein of macromolecular, and there are time-consuming, consumption money, manufacturing cycles for this method Many disadvantages such as long.Immunogene induces high special antibody response, it is necessary to including B cell epitope (or peptide haptens) With Th cell epitope, this be will be apparent from.How to select suitable t helper cell (Th) epitope and how with B cell epitope Or the connection of peptide haptens is a world-famous puzzle come the immunogenicity for significantly improving B cell epitope or peptide haptens, is especially used This immune original production prepares therapeutic vaccine and prepares the single gram of drop antibody or genetic engineering for treating the special hypotype of disease Antibody has huge challenge.

Peptide-carrier albumen coupling is chiefly used in preparing anti-polypeptide antibody, individual polypeptide typically too it is small be not enough to evoke fill The immune response divided, and the carrier protein with many epitopes is conducive to stimulate complementary T cell, further induces B thin Born of the same parents' immune response,.Immune system is by peptide-albumen as a whole come in the antibody that evokes immune response, thus generate Have for polypeptide, have for linking agent, also have for carrier protein, therefore by with B cell epitope or peptide haptens Antibody, which is prepared, with biology immune after carrier protein couplet or fusion is especially used to prepare monoclonal antibody and common phagocytosis now Body antibody library etc. is exactly that prepare genetic engineering antibody be usual common immunological technique, therefore the later period, to generate multiple types anti- The antibody that body needs screening becomes more difficult, carrier keyhole limpet hemocyanin (KLH, the keyhole most generally used Limpet hemocyanin) than bovine serum albumin(BSA) (BSA) there is higher immunogenicity, thus be the carrier being most often selected Albumen.BSA (Bovine Serum Albumin), i.e. bovine serum albumin(BSA), belong to most stable of and soluble albumin, are A kind of popular poor antigen compound carrier albumen.BSA is disadvantageous in that in many experiments, it is taken as closing Agent uses, if polypeptide-BSA conjugate antiserum is in such detection and analysis, it will usually there is false positive, because These serum contain the antibody of anti-BSA.In addition, the antibody generated after KLH and BLH is immune is on the high side with Th1 antibody, for connecting B The immunogene that cell epitope or peptide haptens are formed is to be not suitable for being used to treat the higher disease of Th1 such as type 1 diabetes and concurrent Disease and related disease, atherosclerosis, cardiovascular disease, senile dementia, kidney trouble, ephritis, rheumatoid arthritis, reaction Property arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, erythema nodosum, habitual abortion etc.. Therefore finding suitable carrier molecule and capable of generating the carrier molecule of inclined Th2 antibody is always the important directions probed into.

P277 peptide is one section of specific polypeptide of 437~460 of HSP60, totally 24 amino acid, and can be with effect T The antigenic determinant of cell effect is the specific fragment to play a role in T1DM prevention.DiaPep277 is at present to enter III Phase clinical research.Have experimental studies have found that being carried out according to the identical subcutaneous administration mode of external DiaPep277 with P277 peptide Administration, can cause vascular endothelial cell damage, and induces human body and generate antibody to induce serious atherosclerosis；It will P277 is divided into P1 peptide fragment (437~450) and P2 peptide fragment (448~460) two parts, it has already been proven that P1 polypeptide has certain rush Atherosclerosis, and P2 polypeptide is the Th2 epitope peptide that P277 plays a major role to T1DM, but do not use its as Carrier peptides significantly increase the report of B cell epitope or peptide haptens immunogenicity, do not use especially this total as carrier peptides Valence connects B cell epitope or peptide haptens to prepare energy specificity and epitope containing B cell or the protein binding of peptide haptens and prevent Control the report of the monoclonal antibody of related disease.

Islet cells specific antigen-insulinoma GAP-associated protein GAP (Insulinoma associated protein-2, IA- It 2) is considered as the master for starting autoimmune diabetes i.e. type 1 diabetes (Type 1 diabetes mellitus, T1DM) One of target antigen is wanted, mainly in brain, pancreas, insulinoma is expressed in neuroendocrine cell.The membrane-proximal region JM2's of IA-2 626~630 linear sequences are a B cell epitopes (626FEYQD630), name it for IA2 (5).With IA-2 or its In B cell epitopic immune animal pattern be type 1 diabetes prevention and treatment a strategy, but without be used to do carrier peptides or load A part of body peptide resists to promote the immunogenicity of B cell epitope or peptide haptens and be used to prepare antibody especially monoclonal The report of body.

Summary of the invention

Goal of the invention:

In view of this, one of the objects of the present invention is to provide the carrier peptides for having a kind of composed structure similar and containing this The immunogene of carrier peptides.This carrier peptides epitope containing t helper cell (Th epitope), carrier peptides and B cell epitope or synthetic peptide half are anti- Original is covalently attached and forms peptide based immunogens, can be used for preparing vaccine and multiple types antibody, can be mostly anti-, monoclonal antibody And need with the various genetic engineerings that antibody library or the antibody production techniques such as unicellular preparation are used after this immunogen immune and other Antibody inhibits enzyme activity or GAP-associated protein GAP function, for preventing and treating disease relevant to peptide epitopes and complication and exploitation detection reagent With.Vaccine and multiple types antibody of preparation and combinations thereof, derivative and modification can be used for preventing and treating CD8⁺、Th1 Class cell and cell factor etc. be higher or dominant or a variety of diseases of oxidative stress abnormal induction include type 1 diabetes and simultaneously Send out disease and related disease senile dementia, atherosclerosis and cardiovascular disease etc., kidney trouble, ephritis, rheumatoid joint Inflammation, adjuvant arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, erythema nodosum, habituation stream Produce etc. and for detecting related specific target antigen or main sections.

The second object of the present invention is to provide the technical solution of the carrier peptides and the immunogene preparation containing this carrier peptides.

The present invention relates to one kind to form similar carrier peptides, it is characterised in that the carrier peptides contain a basic polypeptide sequence Column, the polypeptide sequence contain amino acid sequence 1 and amino acid sequence 2, and the amino acid sequence 1 is insulinoma GAP-associated protein GAP A B cell epitope IA2 (5) of IA-2, amino acid sequence (626 FEYQD 630) are shown in SEQ ID NO.1；The amino acid Sequence 2 is the Th2 cell epitope P2 (448 IPALDSLTPANED 460) of the C-terminal of P277 and its changes structure peptide (448 or 455 For any amino acid residue, amino acid sequence is shown in SEQ ID NO.2).455 be amino acid residue A when, be named as PA, 448 be amino acid residue A when, be named as AP

The invention reside in provide the preparation method of this carrier peptides, it is characterised in that the determination of its amino acid sequence is according to such as Lower step is realized:

By the C-terminal of IA2 (5) peptide fragment FEYQD and Th2 epitope P2 and change the N-terminal of structure peptide peptide fragment directly or by flexible peptide or The connection of joining peptide forms a kind of carrier peptides, is named as IA2 (5)-P2 (being directly connected to), passes through flexible peptide or joining peptide Connection be IA-2 (5)-P2-1,2-IA-2 (5)-P2-1,3-IA-2 (5)-P2-1,4-IA-2 (5)-P2-1 (be abbreviated as IP, 2-IP, 3-IP and 4-IP) etc. again with the C-terminal of B cell epitope or synthetic peptide haptens (being indicated with B) directly or by flexible peptide Or link peptide is covalently attached, and forms peptide based immunogens B-IA2 (5)-P2-1, B-2-IA2 (5)-P2-1, B-3-IA2 (5)-P2-1, B-4--IA2 (5)-P2-1 (being abbreviated as B-IP, B-2-IP, B-3-IP and B-4-IP) etc., such carrier peptides in this structure The B cell epitope of connection or the immunogenicity of synthetic peptide based immunogens can be significantly increased, the peptide based immunogens of the peptide containing examples of such carriers can With the mostly anti-, monoclonal antibody for immune biology preparation and need through the immune single-stranded anti-come what is prepared of such peptide based immunogens The genetic engineering antibodies such as body, Fab antibody and other antibody.

Further, the C-terminal and B cell epitope or peptide haptens for being also possible to IA2 (5) peptide fragment FEYQD pass through flexible peptide or After link peptide is covalently attached, then formed by the connection of flexibility peptide or joining peptide with the N-terminal of Th2 epitope P2 peptide fragment novel more Peptide based immunogens IA2 (5)-B-P2-1,2-IA2 (5)-B-P2-1 etc., can also significantly increase B cell epitope or synthetic peptide is immune The immunogenicity of former B.

Further, being to provide to connect the N-terminal of the C-terminal of linear B cell epitope IA2 (5) and P2 peptide to form peptide The flexible peptide of immunogene or the amino acid sequence of joining peptide, or by the C-terminal of IA2 (5) peptide FEYQD and B cell epitope or The ammonia of flexible peptide or joining peptide that the peptide that the N-terminal of peptide haptens B is covalently attached is connected with the N-terminal of Th2 epitope P2 peptide again Base acid sequence, or the N-terminal of the N-terminal and B cell epitope IA2 (5) of B cell epitope or peptide haptens B is covalently attached Form the flexible peptide of polypeptide or the amino acid sequence of joining peptide.

Further, the upper flexible peptide or connection peptide linker are X1X2X3, --- --- Xn, --- --, X1X2X3X4X5, X1X2X3X4, X1X2X3, X1X2, X, GGGGG, GGGG, GGG, GGS, GG, G, K, KK or other small peptides.X1X2X3X4, X1X2X3X4X5, X1X2X3X4X5X6 and X1X2X3------Xn, the amino acid sequence are shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.Xn is any amino acid residue.The preferred 1-20 Arabic numerals of n.

The third object of the present invention is to provide and be resisted with such carrier peptides by the target that flexible peptide or joining peptide connect Former B cell epitope or synthesis haptens, these B cell epitopes or synthesis haptens and routine carrier B SA or KLH coupling are exempted from It can produce the immune serum (antibody containing Th1 and Th2 type, Th1 are slightly more) greater than 10000 potency after epidemic disease, and it is of the invention Such carrier peptides can be as B cell epitope or the carrier and immunologic stimulant of synthetic peptide haptens (B), its significant enhancing is exempted from Epidemic focus, can also generate the antibody titer requirement that all kinds of antibody are used to prepare greater than 10000 potency, the antibody energy of preparation and contain B The specific antigen or immunogene of cell epitope or synthetic peptide based immunogens combine and Th2 type antibody is dominant.It therefore can be containing load On the basis of the immunogen immune of body peptide, takes the spleen bone-marrow-derived lymphocyte that biology is immunized to be directed to by antibody production techniques preparation and contain The specific antibody of the target antigen of this B cell epitope or synthetic peptide haptens (B), including mostly anti-, monoclonal antibody, it is single-stranded anti- The genetic engineering antibodies such as body and Fab antibody.

The fourth object of the present invention is that the carrier peptides provided and the peptide based immunogens containing the carrier peptides can be used for weight Group microorganism and transgenic animals or plant and its cell, make them as production plant, are able to produce the peptide containing this carrier peptides Immunogene and its fusion protein, coupling protein or conjugate, polymer, composition or production oral vaccine are produced based on this Inhibit target antigen after specific antibody and its derivative and analogue or small molecular antibody, immunogen immune or antibody administration The activity of albumen, significantly adjusting CD4+/CD8+, Th1/Th2 dysequilibrium are biased to Th2 transfer by Th1, increase CD4+, lower CD8+T cell adjusts CD4+/CD8+ and tends to range of normal value, and significantly improves the disease symptom of the higher related disease of Th1.

The fifth object of the present invention is to provide comprising including the carrier peptides and peptide based immunogens described in immunological effective amount And its fusion protein, coupling protein, polymer, composition and recombinant microorganism and transgenic animals or plant and its cell.

Further, also comprising pharmaceutically acceptable carrier and adjuvant or pharmaceutically acceptable auxiliary material.The peptide Immunogene and peptide vaccine and pharmaceutically active substance or pharmaceutical carrier be made fusion protein, coupling protein, polymer, composition and The application of recombinant microorganism and transgenic animals or plant and its cell.

The pharmaceutically acceptable pharmaceutically active substance, including but not limited to: GLP-1, insulin, biguanides, sulphur Ureas；Wherein biguanides include insoral, buformin, melbine；Sulfonylurea drugs include column phenylurea, Mick pancreas, lattice column Qi Te, gram sugared benefit, Glibornuride, gliquidone, Gliquidone, mindiab, Gliguidone etc..

Further, pharmaceutically acceptable carrier, including but not limited to: heat shock protein HSP60/65, asparagine Enzyme, L-Asparaginasum-TTP, L-Asparaginasum-TTP-CETPC, Fc, bovine serum albumin(BSA) (BSA), keyhole limpet hemocyanin (KLH), polyethylene glycol etc..

Further, the pharmaceutically acceptable auxiliary material: for filler, diluent, excipient, adjuvant etc..For example including But it is not limited to: Freund's adjuvant, aluminium adjuvant, lactose, sucrose, glucose, starch, cellulose family (such as carboxymethyl cellulose, hydroxypropyl Methylcellulose etc.), ethylene glycol, soya-bean oil, sesame oil, ethyl alcohol, sterile saline, sterile water, mannitol adds Lipofundin Lipofundin and all kinds of immunogenes or the adjuvant of antigen etc..

The auxiliary material is filler, diluent, excipient etc..For example including but be not limited to: lactose, sucrose, grape Sugar, starch, cellulose family (such as carboxymethyl cellulose, hypromellose), ethylene glycol, soya-bean oil, sesame oil, ethyl alcohol, nothing Bacterium physiological saline, sterile water etc..

The beneficial effects of the present invention are:

Biology, which is immunized, in peptide based immunogens containing such carrier peptides can significantly improve the B cell epitope in target antigen site Or it synthesizes the immunogenicity of haptens and significantly improves the amount of antibody especially Th2 subclass antibodies, the peptide containing such carrier peptides Immunogene can be used for preparing it is special with epitope containing the B cell or peptide haptens protein bound specific antibody of target antigen, It can inhibit the enzymatic activity or protein function of target protein, it is significant to adjust CD4+/CD8+ and Th1/Th2 dysequilibrium, by Th1 It is biased to Th2 transfer, increases CD4+, lowers CD8+T cell, significantly improves the disease symptom of disease.And containing the carrier peptides and Its derivative and analogue and coupling protein and fusion protein and its immune specific antibody and antibody derivatives generated or prepare For recombinant microorganism and turn with the pharmaceutical preparation of analog, and the carrier peptides provided and peptide based immunogens containing this carrier peptides Genetic animal or plant and its cell make them as production plant, produce peptide based immunogens containing carrier peptides and fusion protein and Coupling protein, composition or production oral vaccine or the pharmaceutical preparation for producing specific antibody and its derivative and analogue, pre- Anti- and treatment CD4+ lowers and CD8+T cell increases and the disease rheumatism of Th1 dominance induction, multiple sclerosis, glycosuria Disease, nephrosis, atherosclerosis, hyperlipidemia, high lithemia, senile dementia, eye disease, pedopathy, angiocarpy etc. and CD8+T are thin Born of the same parents increase disease such as type 1 diabetes, systemic loupus erythematosus, rheumatoid arthritis, chronic active hepatitis, infectiousness list Nucleus increase disease and other virus infections and infectious disease etc. have significant application value, by immune containing this carrier peptides The immune specific antibody for generating and being prepared by various technologies of original and its derivative and analogue or modification are for prevention and treatment phase Related disorders or detection related objective antigen protein, are developed as drug or detection reagent.

Further, when B cell epitope or synthesis haptens selection are 4 (dipeptidyl of people's dipeptidyl peptidase Peptidase 4, DPP4) advantage B cell antigen epi-position or synthetic peptide haptens, the N-terminal of itself and carrier peptides is passed through into flexibility Peptide or link peptide are connected to form peptide based immunogens D41-IP, D41-IPA, D41-IAP, D41-2-IP, D41-3-IP, D41-4- IP and D41-I6P etc. is coupled among IA2 (5) and P2, forms the peptide based immunogens such as IA2 (5)-D41-P2-1, immune High-level antibody can be generated after normal mouse, but does not cause hypoglycemia, and there is safety；In the diabetes of immune STZ induction With can be generated after NOD diabetic mice specificity the antibody (belonging to Th2 hypotype) with DPP4 ining conjunction with, inhibition enzyme activity or GAP-associated protein GAP function can be used for detecting specific antigen, and antibody titer is mainly distributed between 8000-20000, increase Th2 class Cell factor adjusts Th1/Th2 balance, increases Th2 and GLP-1 amount, increase amount of insulin, protect islet cells, it is high to reduce pancreas Blood glucose element reduces blood glucose, and uric acid creatinine improves oxidative stress, increases CD4+, lowers CD8+T cell, can be treatment and DPP4 Exception, pathoglycemia, dysglycemia and the unbalance caused diabetes of Th1/Th2, nephrosis, atherosclerosis, old age are silly The diseases such as slow-witted, eye disease, pedopathy, angiocarpy provide new way；The novel polypeptide immunogene of the B cell antigen epi-position of advantage containing DPP4 Prevention and treatment diabetes, including type 1 diabetes, diabetes B, the classes patients with type Ⅰ DM such as gestational diabetes can be developed.It can also use In prepare antibody especially Th2 antibody for treatment related disease or detection.Animal is immunized with the peptide based immunogens containing carrier peptides, Th2 subclass antibodies IgG1 and IgG2b are belonged to by monoclonal antibody prepared by hybridoma technology again, administration prevents and treats sugar Urinate disease model mouse, control and reduce animal blood glucose, can specificity with DPP4 enzyme ining conjunction with, increase Th2 type cytokines, tune Th1/Th2 balance is saved, DPP4 enzymatic activity is reduced, reduces serum uric acid and creatinine amount, increase CD4+, lower CD8+T cell, protect Protect the tissue such as pancreas and kidney.Potency, which meets the requirements, after immunogen immune can be used for preparing antibody and passes through monoclonal antibody The antibody that Th2 IgG antibody 1 (mouse) or human IgG2 a (people) preparation are prepared with the technologies such as genetic engineering, can also treat CD4 +/CD8+ and Th1/Th2 dysequilibrium related disease or detection are used, and CD4+ is increased, and lower CD8+T cell, are improved Th2 and are lowered The Th1 class T cell factor, significantly improves the disease symptom of the higher disease of Th1.

Further, when B cell epitope or synthesis haptens selection are people's xanthine oxidoreductase enzyme (xanhtine Oxidoreductase, XOD) advantage B cell antigen epi-position or synthetic peptide haptens, the N-terminal of itself and such carrier peptides is led to It crosses flexible peptide or link peptide is connected to form peptide based immunogens O-IP, O-IPA, O-IAP, O-2-IP, O-3-IP, O2-IP and O- The polypeptide immunogens such as I6P, or be coupled among IA2 (5) and P2, IA2 (5)-O-P2-1 is formed, it is same after immune mouse Sample can generate the antibody (belonging to Th2 hypotype) in conjunction with xanthine oxidase of specificity, increase Th2 type cytokines, adjust Th1/Th2 balance, increases amount of insulin, protects islet cells, reduces blood glucose, reduces xanthine oxidase activity, reduces uric acid Creatinine, increasing significantly increase oxidation resistance.The vaccine and antibody for the treatment of diabetic complication can be developed.To be contained containing such Animal is immunized in the peptide based immunogens of body peptide, can be used for preparing antibody and by the preparation of the technologies such as monoclonal antibody and genetic engineering Th2 IgG antibody 1 (mouse) or human IgG2 a (people) are for treatment CD4+/CD8+ and Th1/Th2 dysequilibrium related disease or detection With increase CD4+ lowers CD8+T cell, improves Th2 and simultaneously lowers Th1 class T cell and antibody, significantly improves the morbidity disease of disease Shape.Drug treatment high lithemia model mouse, does not reduce animal blood glucose, can specificity with xanthine oxidase ining conjunction with, raising Th2 type cytokines adjust Th1/Th2 balance, reduce xanthine oxidase activity, reduce serum uric acid and creatinine amount, increase Add uric acid and creatinine excretion, increase CD4+, lower CD8+T cell, protects kidney.It can develop that be used to prepare antibody and Th2 anti- Body such as IgG1 is for treatment related disease or detection.

Further, when B cell epitope or synthesis haptens selection are people's xanthine oxidoreductase enzyme (xanhtine Oxidoreductase, XOD) advantage B cell antigen epi-position or synthetic peptide haptens, by its N-terminal with the polypeptide directly or It is connected to form novel polypeptide immunogene O-IA2 (5)-P2, O-IA2 (5)-P2-1, O2-IA2 by flexible peptide or link peptide (5) polypeptide immunogens such as-P2-2, or be coupled among IA2 (5) and P2, IA2 (5)-O-P2 is formed, after immune mouse The antibody (belonging to Th2 hypotype) in conjunction with xanthine oxidase that specificity can be generated, increases Th2 type cytokines, adjusts Th1/Th2 balance, increases amount of insulin, protects islet cells, reduces blood glucose, reduces xanthine oxidase activity, increases significant increase Strong anti-oxidation ability.The vaccine and antibody for the treatment of diabetic complication can be developed.Animal is immunized with peptide based immunogens, then passes through The monoclonal antibody of hybridoma technology preparation belongs to Th2 hypotype, peptide based immunogens and the high urine of antibody administration treatment based on its preparation Acid profile mouse, does not reduce animal blood glucose, can specificity with xanthine oxidase ining conjunction with, increase Th2 type cytokines, tune Th1/Th2 balance is saved, xanthine oxidase activity is reduced, reduces serum uric acid and creatinine amount, increase uric acid and creatinine excretion, Reduce renal inflammation.It can develop and be used to prepare Th2 antibody for treatment related disease or detection.

Further, when B cell epitope or synthesis haptens be selected as people's urate transporter 1 (URAT1) advantage B it is thin It is connected to form novel polypeptide immunogene UIP- by extracellular antigen epitope with the N-terminal of the carrier peptides by flexible peptide or link peptide 1 (U1-IP), can generate the specific IgG antibody 1 in conjunction with URAT1 after immune mouse and IgG2b (it is sub- to belong to Th2 Type), it improves Th2 and lowers Th1 class T cell and antibody, increase Th2 type cytokines, adjust Th1/Th2 balance, increase pancreas islet Element amount, protects islet cells, reduces blood glucose, reduces serum uric acid and creatinine, improves anti-oxidation stress ability, protection pancreas and Kidney.Equally immune biology can be used for preparing antibody and prepare Th2 antibody by technologies such as monoclonal antibody and genetic engineerings IgG1 (mouse is IgG2a in people Th2) increases for treatment CD4+/CD8+ and Th1/Th2 dysequilibrium related disease or detection Add CD4+, lower CD8+T cell, improve Th2 and lower Th1 class T cell and antibody, significantly improves the disease symptom of disease., With most important theories value and broad prospect of application, there is important social and economic benefit.

Further, when the prediction B cell epitope of selection target antigen albumen human acetylcholinesteraseisomer or synthetic peptide half are anti- It is connected to form peptide based immunogens A1-IP, A2-IP, exempted from by original with the N-terminal of such carrier peptides by flexible peptide or link peptide Also the potency that specificity can be generated after epidemic disease mouse reaches 16000 antibody, can develop and be used to prepare antibody and Th2 IgG antibody 1 (mouse) or human IgG2 a (people) increase CD4 for treatment CD4+/CD8+ and Th1/Th2 dysequilibrium related disease or detection +, lower CD8+T cell, improves Th2 and lower Th1 class T cell and antibody, significantly improve the disease symptom of related disease.

Further, when the advantage B cellular antigens of selection and tumor correlated albumen people programmed death receptors ligand PD-L1 Epitope or synthetic peptide haptens, it, which is connected by flexible peptide or link peptide with the N-terminal of such carrier peptides, to form peptide and is immunized Original can generate potency in 12000 or so specific antibody (it is dominant to belong to Th2 hypotype) after immune mouse, can also drop Hypoglycemia improves diabetic symptom.It equally can be used for exploitation and prepare antibody and Th2 IgG antibody 1 (mouse) or human IgG2 a (people) increases CD4+ for treatment CD4+/CD8+ and Th1/Th2 dysequilibrium related disease or detection, lowers CD8+T cell, It improves Th2 and lowers Th1 class T cell and antibody, significantly improve the disease symptom of related disease

When the advantage B cell antigen epi-position or synthetic peptide haptens for selecting any one other target antigen albumen such as The B epitope or synthetic peptide haptens of influenza neuraminidase antigen, by the N-terminal of itself and the carrier peptides pass through flexible peptide or Link peptide is connected to form peptide based immunogens, and the antibody that the specificity of potency 12000 or so can be generated after immune mouse (belongs to It is dominant in Th2 hypotype), blood glucose is reduced, improves diabetic symptom, can develop and be used to prepare antibody and Th2 IgG antibody 1 (mouse) or human IgG2 a (people) increase CD4 for treatment CD4+/CD8+ and Th1/Th2 dysequilibrium related disease or detection +, lower CD8+T cell, improves Th2 and lower Th1 class T cell and antibody, significantly improve the disease symptom of related disease.Tool There are most important theories value and broad prospect of application, there is important social and economic benefit.

Detailed description of the invention

In order to make the object of the invention, technical solution and advantage understand, are further retouched in detail in conjunction with attached drawing to the present invention It states, in which:

Fig. 1 is after C57BL/6J hero mouse is immunized respectively in peptide based immunogens D41-IP, O-IP, U-IP containing carrier peptides of the present invention Serum generate antibody respectively with people DPP4 (Figure 1A, Lane 1:marker；Lane 2: recombined human DPP4 holoenzyme；Lane 3: D41-BSA；Lane 4:BSA；Lane 5:L-ASP irrelevant protein) and cow-bezoar purine oxidase segment (Figure 1B, 1: marker；Lane 2: cow-bezoar purine oxidase segment；Lane 3:BSA；Lane4:L-ASP irrelevant protein) and uric acid transporter Albumen 1 (Fig. 1 C, 1:marker；Lane 2: urate transporter 1；Lane 3:BSA；Lane4:L-ASP irrelevant protein) it is special The Western Blot experimental result picture of different combination；

Fig. 2 is that normal Balb/c mouse is immunized in peptide based immunogens D41-IP, O-IP, U-IP containing carrier peptides of the present invention respectively Afterwards, the spleen bone-marrow-derived lymphocyte of adaptive immune, (D41-IP exempts from the monoclonal antibody CPU-KD4 prepared by hybridoma technology The monoclonal antibody prepared after epidemic disease), CPU-KO1 (monoclonal antibody for preparing after O-IP is immune), CPU-KU1 is (after U-IP is immune The monoclonal antibody of preparation) respectively with people DPP4 (Fig. 2A, Lane 1:marker；Lane 2: recombined human DPP4 enzyme；Lane 3: D41-BSA；Lane 4:BSA；Lane 5:L-ASP irrelevant protein) and cow-bezoar purine oxidase segment (Fig. 2 B, 1: marker；Lane 2: cow-bezoar purine oxidase segment；Lane 3:O-BSA；Lane 4:BSA；Lane5:L-ASP unrelated protein Matter) and urate transporter 1 (Fig. 2 C, 1:marker；Lane2:U-BSA；Lane3:BSA；Lane4:L-ASP unrelated protein Matter；Lane 5: urate transporter 1) specific bond Western Blot experimental result picture；

Specific embodiment

Embodiment 1: carrier peptides IA-2 (5)-P2-1,2- of the epitope containing t helper cell (Th epitope) of a kind of composed structure IA-2 (5)-P2-1,3-IA-2 (5)-P2-1,4-IA-2 (5)-P2-1, IA-2 (5) -6-P2-1 etc. (is abbreviated as IP, 2-IP, 3- IP, 4-IP, I6P etc.) preparation.

Linear B cell epitope IA2 (5) peptide fragment of IA-2 is selected, amino acid sequence 1 is shown in SEQ ID NO.1, P277's The Th2 epitope P2 peptide fragment of C-terminal, amino acid sequence 2 is shown in the structure peptide that changes of SEQ ID NO.2, P2, and (448 or 455 is any Amino acid residue, amino acid sequence are shown in SEQ ID NO.2).455 when being amino acid residue A, are named as PA, 448 are ammonia When base acid residue A, it is named as AP.

Modification is analyzed and identified with the screening of a variety of analysis softwares and the experiment of multiple STZ diabetic rat model, is determined two tables Position is connected with flexible peptide, and the C-terminal of IA2 (5) peptide fragment FEYQD and the N-terminal of Th2 epitope P2 peptide fragment are passed through different flexible peptide or company The connection for connecing peptide fragment forms carrier peptides IP, IPA, IAP, 2-IP, 3-IP, 4-IP, 6-IP and I6P etc., flexible peptide or link peptide ammonia Base acid sequence is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.IP, IPA, IAP, 2- IP, 3-IP, 4-IP, 6-IP and I6P, amino acid sequence see respectively SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14；Or by IA2 (5) After the C-terminal and B cell epitope or peptide haptens of peptide fragment FEYQD passes through flexible peptide or link peptide is covalently attached, then with Th2 epitope The N-terminal of P2 peptide fragment forms peptide based immunogens IA2 (5)-B-P2-1 etc., IA2 (5)-D41- by the connection of flexible peptide or joining peptide The amino acid sequence of P2-1 and IA2 (5)-O-P2-1 are shown in SEQ ID NO.15 and SEQ ID NO.16 respectively.

The preparation of embodiment 2:B cell epitope or synthetic peptide haptens B.

Software and on-line analysis tool are analyzed with a variety of B cell antigen epi-positions, based on one-parameter, in conjunction with two Level structure prediction and space structure feature, from a target antigen protein, (such as: people DPP4 or people XOD or human urine acid turn respectively Fortune albumen 1 or human acetylcholinesteraseisomer) more than 100 a epitopes in select multiple advantage B cell antigen epi-positions or peptide haptens B, By being connected with carrier peptides IP, 2-IP, 3-IP, 4-IP, 6-IP and I6P etc. with flexible peptide, by immune zoopery screening and Further conversion and the process screened again, the high B cell antigen epi-position of adaptive immune originality or synthetic peptide haptens B.

Embodiment 2-1 is when the advantage B cell that B is people's dipeptidyl peptidase 4 (dipeptidyl peptidase 4, DPP4) Epitope, preparation method are that D41, D42, D43 are selected from its more than 100 a epitope, the multiple prediction advantages of D44----- etc. B cell antigen epi-position or peptide haptens B, by the N-terminal with carrier peptides IP, 2-IP, 3-IP, 4-IP, 6-IP and I6P etc. with soft Property peptide connect to form peptide based immunogens, by the high B cell antigen epi-position or peptide of zoopery screening adaptive immune originality half is immunized Antigen D41, D42, D43 etc..D41 is a segment polypeptide sequence 88VFLENSTFDE97 of DPP4, totally 10 amino acid residues.

Embodiment 2-2 is as the advantage B that B is people's xanthine oxidoreductase enzyme (xanhtine oxidoreductase, XOD) Cell antigen epitope selects 0,01,02,03----- etc. multiple prediction advantage B cell antigen tables from its more than 100 a epitope Position or peptide haptens B, by connecting to be formed with flexible peptide with the N-terminal of I6P with carrier peptides IP, 2-IP, 3-IP and 4-IP, 6-IP Peptide based immunogens, by zoopery screening and further conversion being immunized and the process screened, the high B cell of adaptive immune originality are anti-again Former epitope or peptide haptens 0,02 etc..0 is a segment polypeptide sequence of xanthine oxidoreductase enzyme 1101RLEPYKKKNPS1111, totally 11 amino acid residues.

Embodiment 2-3 when B be people's urate transporter 1 (URAT1) prediction advantage B cell antigen epi-position, from its 100 U, U1, U2, the multiple advantage B cell antigen epi-positions of U3----- etc. or peptide haptens X are selected in multiple epitopes, by with carrier Peptide IP etc. is anti-by the high B cell antigen epi-position or peptide half of zoopery screening adaptive immune originality is immunized with the connection of flexible peptide Former U.

Embodiment 2-4 is when the prediction advantage B cell antigen that B is with senile dementia related antigen such as human acetylcholinesteraseisomer Epitope selects B1, the multiple advantage B cell antigen epi-positions of B2, B3----- etc. or peptide haptens B from its more than 100 a epitope, By connecting to form peptide based immunogens with flexible peptide with IP etc., by the high B cell of zoopery screening adaptive immune originality is immunized Epitope or peptide haptens A1, A2.

Embodiment 2-5 is thin as the prediction advantage B that B is other diseases associated antigen protein such as tumor associated target mark PDL-1 Extracellular antigen epitope selects LDP1, the multiple advantage B cell antigen epi-positions of LDP2, LDP3----- etc. from its more than 100 a epitope Or synthetic peptide peptide haptens B, by connecting to form peptide based immunogens with flexible peptide with IP etc., by immune zoopery screening It can be with the high B cell antigen epi-position of adaptive immune originality or peptide haptens LDP2.

Embodiment 3: peptide based immunogens B-IP, B-2-IP, B-3-IP and the B-4-IP containing carrier peptides, B-6-IP, B-I6P, The preparation and synthesis of IA2 (5)-B-P2-1 (B is B cell epitope or synthetic peptide haptens).

Software and on-line analysis tool are analyzed with B cell antigen epi-position, based on multiple one-parameters, in conjunction with two Level structure prediction and space structure feature, (such as: people DPP4 or XOD or urate transporter 1 from a target antigen protein Or acetylcholinesterase etc.) more than 100 a epitopes in select multiple advantage B cell antigen epi-positions or peptide haptens B, by the C of B End is connected with the N-terminal of above-mentioned carrier peptides, peptide based immunogens is constituted, when B is people DPP4, xanthine oxidase, uric acid transporter egg respectively White 1 and acetylcholinesterase advantage B cell epitope or synthetic peptide haptens when respectively constitute peptide based immunogens D41-IP (amino Acid sequence is shown in SEQ ID NO.17), D41-2-IP (amino acid sequence is shown in SEQ ID NO.18), D41-3-IP (amino acid sequence See SEQ ID NO.19), D41-4-IP (amino acid sequence is shown in SEQ ID NO.20), (amino acid sequence is shown in SEQ to D41-6-IP ID NO.21) O-IP (amino acid sequence is shown in SEQ ID NO.22), O-2-IP (amino acid sequence is shown in SEQ ID NO.23), O- 4-IP (amino acid sequence is shown in SEQ ID NO.24), D41-IPA (amino acid sequence is shown in SEQ ID NO.25), D41-IAP (ammonia Base acid sequence is shown in SEQ ID NO.26), O-IPA (amino acid sequence is shown in SEQ ID NO.27), O-IAP (be shown in by amino acid sequence SEQ ID NO.28), IA2 (5)-O-P2-1 (amino acid sequence is shown in SEQ ID NO.16), IA2 (5)-D41-P2-1 (amino acid Sequence is shown in SEQ ID NO.15), U-IP (or UIP-1), influenza neuraminidase B cell epitope and IP are coupled peptide A1- IP, A2-IP, people's programmed death receptors ligand B cell epitope and IP are coupled peptide PD-L1PDL1-IP.

Above-mentioned peptide based immunogens are all made of the synthesis of FMOC solid-phase synthesis, and HPLC detects purity > 90%.It analyzes thin with B Born of the same parents' epitope or synthetic peptide haptens are synthesized using FMOC solid-phase synthesis, and HPLC detects purity > 95%, B cell epitope or conjunction It is coupled again with conventional carriers albumen KLH or BSA at peptide haptens and to form B-BSA, B-KLH (B is B cell epitope or synthetic peptide half Antigen) such as D41-KLH, O-KLH or U-BSA.

Experimental method in embodiment is unless otherwise instructed conventional method.Used kit is produced according to manufacture Condition proposed by product manufacturer carries out.

Embodiment 4: peptide based immunogens B-IP, B-2-IP, B-3-IP and the B-4-IP containing carrier peptides of the present invention, B-, 6-IP, (B is the advantage B of people DPP4 or XOD or urate transporter 1 or acetylcholinesterase etc. to B-I6P, IA2 (5)-B-P2-1 respectively Cell epitope or peptide haptens) i.e. D41-IP, D41-IPA, D41-IAP, D41-2-IP, D41-3-IP, D41-4-IP, D41- 6-IP、O-IP、O-IPA、O-IAP、O-2-IP、 O-4-IP、U-IP、A1-IP、A2-IP、PDL2-IP、IA2(5)-O-P2-1、 IA2 (5)-D41--P2-1, D41-I6P, O-I6P) immune STZ induction diabetic rat model and normal Balb/c mouse Immunogenicity and pharmacodynamic evaluation.

The foundation of the diabetic rat model of embodiment 4-1.STZ induction

Streptozotocin (STZ) inducing mouse diabetes (DM) model is injected intraperitoneally using low dose of continuous several times.4 weeks Age male C 57 BL/6 J mouse (is purchased from Yangzhou University's comparative medicine center, credit number: SCXR (Soviet Union) 2012-0004.) use It is 7.5 mg/ml that 0.1M pH4.4 citrate buffer, which configures STZ concentration, is injected intraperitoneally by 50mg/kg dosage, one time a day, Continuous injection 5 times, the 3rd, 7, the 14 day measurement blood glucose value after modeling, with fasting blood-glucose twice in succession >=11.1mmol/L at Mould standard.

The experiment of embodiment 4-2. animal immune

The immunization experiment of the successful C57BL/6J mouse of embodiment 4-2-1. modeling

240 diabetic mices are randomly divided into 24 groups, every group 10, are respectively as follows: placebo group (solvent control Group, negative control group), P277, IP control group, D41-IP, D41-IPA, D41-IAP, D41-2-IP, D41-3-IP, D41-4- IP、D41-6-IP、O-IP、O-IPA、O-IAP、O2-IP、 O-2-IP、O-4-IP、U-IP、A1-IP、A2-IP、PDL2-IP、 IA2 (5)-O-P2-1, IA2 (5)-D41--P2-1, D41-I6P, O-I6P administration group.Administration group drug concentration is 1mg/ml, Dosage is 100 μ g//times, i.e. 100 μ l medical fluids；Negative control group: 100 μ l finishes/only/time.Medicine ordinance method is pressed 1mg+40mg mannitol be dissolved in the ratio provisional configuration finish of 1ml20%Lipofundin model success (with blood glucose >= Subject to 11.1mmol/L) start within the 2nd week to carry out animal immune afterwards.Weekly administration 1 time, continuous 5 times, then respectively at 7,9,11 weeks It is immunized three times, amounts to 8 times.

The immunization experiment of the normal Balb/c mouse of embodiment 4-2-2.

The normal BALB/c female mice of 4 week old (is purchased from Yangzhou University's comparative medicine center, credit number: SCXR (Soviet Union) 2012-0004.) adaptive feeding is to 7 week old, peptide based immunogens D41-IP, O-IP, U-IP, D41-KLH, the O- that will have been dispensed KLH, U1-BSA, KLH, BSA powder are dissolved in injection physiological saline, are made into 1mg/mL solution；By above-mentioned solution and immune assistant Agent (QuickAntibody-Mouse5W, Beijing Bo Aolong) mixed in equal amounts is placed on vortex mixed instrument and shakes uniformly, is used for flesh Meat drug administration by injection (0.1mL/ is only), interval carries out second with same method after two weeks and is immunized, then is spaced two weeks and takes hematometry small Mouse serum antibody titer.

Peptide based immunogens D41-IP, D41-IPA of the embodiment 4-3. containing carrier peptides of the present invention, D41-IAP, D41-2-IP, D41-3-IP、D41-4-IP、D41-6-IP、O-IP、O-IPA、O-IAP、O2-IP、 O-2-IP、O-4-IP、U-IP、A1-IP、 It is male that C57BL/6J is immunized in A2-IP, PDL2-IP, IA2 (5)-O-P2-1, IA2 (5)-D41--P2-1, D41-I6P, O-I6P respectively The ELISA and antibody titer testing result for the specific antibody that serum generates after mouse and normal BALB/c female mice:

It is coated with dilution (0.05M sodium carbonate-bicarbonate buffer, pH9.6) and dilutes D41-BSA, be made into final concentration of The solution of 0.5mg/mL, 0.1mL is added in every hole in 96 hole elisa Plates, and 4 DEG C of coatings are overnight；Coating buffer is discarded, every hole is added 5% Skimmed milk power (pH7.4PBS preparation) confining liquid, 4 DEG C of full hole closings are overnight；Confining liquid is discarded, 1: 100 (C57BL/ is added in every hole 6J) or 1: 500 (BALB/c), the diluted peptide based immunogens of 5% skimmed milk power immune mice serum 0.1mL, 37 DEG C of incubation 1h； Serum is discarded, every hole is washed with PBST (PBS solution containing 0.5% Tween-20) and distillation water spacer, and each 3min is washed altogether 6 times；After washing, the goat anti-mouse igg secondary antibody 0.1mL of the diluted horseradish peroxidase label of 5% skimmed milk power is added in every hole, 1: 5000,37 DEG C of incubation 1h of extension rate；Secondary antibody liquid is discarded, every hole is washed with PBST and distillation water spacer, each 3min, altogether Washing 6 times；After washing, every hole is added 0.1mL TMB developing solution, and after 37 DEG C of reaction 30min, the H of 2mol/L is added₂SO₄0.1mL Terminate reaction；Distilled water returns to zero, and the OD value in each hole is measured under microplate reader 450nm wavelength.Experimental result is as shown in table 1, and mouse exists Give D41-IP, D41-IPA, D41-IAP, D41-2-IP, D41-3-IP, D41-4-IP, D41-6-IP, O-IP, O-IPA, O- IAP、O2-IP、 O-2-IP、O-4-IP、U-IP、A1-IP、A2-IP、PDL2-IP、IA2(5)-O-P2-1、IA2(5) -D41-- After P2-1, D41-I6P, O-I6P are immune, serum ELISA the results are shown in Table 1 display, generate respectively in serum for people DPP4 or The high-level specific antibody of XOD or urate transporter 1 or acetylcholinesterase etc., has conspicuousness poor compared with the control group Different (P < 0.05), significant difference (P < 0.01) or extremely significant difference (P < 0.001), potency reaches 6000 to 20000, peptide Immunogen immune animal can be used for preparing therapeutic and detection with more anti-, monoclonal antibodies and need immunogen immune ability The genetic engineering antibody of preparation.Normal Balb/c mouse is immunized in D41-IP, O-IP, U-IP, D41-KLH, O-KLH, U1-KLH High-caliber specific antibody is generated, potency has extremely significant difference (P < 0.001) compared with the control group,

1 peptide epitopes of table and peptide based immunogens immune serum antibody ELISA and bioactivity result containing carrier peptides

P compared with IP ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05；

It is male that C57BL/6J is immunized in embodiment 4-4. peptide based immunogens D41-IP, O-IP, U-IP containing carrier peptides of the present invention respectively Serum generates Western Blot experiment of the antibody in conjunction with antigen-specific after mouse:

Serum and people DPP4 Western Blot experimental basis Western Blot standard practice instructions carry out, by DPP4 It is transferred on pvdf membrane, jump condition 100V, 5h；Pvdf membrane after transfer as shaking at room temperature in 25mL Block buffer Dynamic 1h.15mL TBST is washed 3 times (5min/ times)；Then pvdf membrane is soaked in 1: 10 diluted Antigenic Peptide immune serum In, it is incubated at room temperature 1-2h or 4 DEG C overnight, slowly shakes, 15mL TBST is washed 3 times (5min/ times).It is diluted to add 1: 5000 The sheep anti-mouse igg of horseradish peroxidase-labeled, 37 DEG C of incubation 1h.PBST washes film 3 times, each 10min.DAB developing solution is added It develops the color.Sufficiently by film transfer, into deionized water, color development stopping is reacted after colour developing.Antigen is after SDS-PAGE electrophoresis, directly It connects in electrotransformation to pvdf membrane, after closing, the serum obtained with peptide based immunogens D41-IP (or O-IP or U-IP) immunized mice is one Anti-, sheep anti-mouse igg is that secondary antibody carries out immuno absorbence to not synantigen 4 respectively, and experimental result on film as shown in Figure 1, occur special Property the purpose band that combines of serum and people DPP4 (or people's xanthine oxidase or urate transporter 1), and egg unrelated with other It is white to be not bound with, it can be used for detecting specific antigen albumen.

Balb/c mouse is immunized in embodiment 4-5. peptide based immunogens D41-IP, O-IP, U-IP containing carrier peptides of the present invention respectively The detection of serum antibody hypotype afterwards:

The assay kit of subtype-specific antibody is purchased from Mei Mian biotech firm, and experimental method carries out to specifications.Greatly The experiment flow of cause is as follows: 1,50ul standard items or 5 times of diluted serum are added in every hole.2,100ul enzyme mark is added in every hole Secondary antibody, 37 DEG C of incubation 60min after preservative film winding.3, wash solution expires hole board-washing 5 times.4,50ul substrate is added in every hole A and 50ul substrate B, mixes gently, and 37 DEG C are protected from light incubation 15min.5,50ul terminate liquid (2M H is added in every hole₂SO₄) (color by Indigo plant turns yellow).6, OD value is measured in 15min at 450nm wavelength.7, using standard items OD value as abscissa, concentration value is vertical sits Mark draws standard curve, calculates sample concentration.Experimental result is as shown in table 3, the results showed that D41-IP, O-IP, U-IP are immune The specificity T h2 type IgG antibody 1 generated in serum after Balb/c mouse, is significantly higher than IP and solvent group, has statistics poor Different (P < 0.01).IgG2b is also significantly increased.It is produced in serum after peptide epitopes and the immune Balb/c mouse of KLH or BSA covalent linkage Raw specificity T h1 type IgG antibody 2a, it is significant to increase and be higher than control group, and 1 elevation amplitude of Th2 type IgG antibody is not so good as Th1 The immune animal connection different carriers of type IgG antibody 2a, peptide epitopes and peptide based immunogens may be incorporated for the more anti-, monoclonals of preparation and resist Body and genetic engineering antibody, but prepare the treatment higher disease of Th1 and need carrier peptides of the invention.Peptide epitopes can be used for making Standby Th2 and Th1 antibody, for treating and detecting the antigen protein containing epitope peptide.

The testing result of serum antibody hypotype after mouse is immunized in 3 peptide epitopes of table and peptide based immunogens

P compared with IP ^*P < 0.05,^**P < 0.01；^#P < 0.05,^##P < 0.01；^&P < 0.01,^&&P < 0.01

Peptide based immunogens D41-IP, D41-IPA of the embodiment 4-6. containing carrier peptides of the present invention, D41-IAP, D41-2-IP, D41-3-IP、D41-4-IP、D41-6-IP、O-IP、O-IPA、O-IAP、O2-IP、O-2-IP、O-4-IP、U-IP、A1-IP、 It is male that C57BL/6J is immunized in A2-IP, PDL2-IP, IA2 (5)-O-P2-1, IA2 (5)-D41--P2-1, D41-I6P, O-I6P respectively Hypoglycemic experiment after mouse:

In model success (be subject to blood glucose >=11.1mmol/L) weekly administration 1 time afterwards, continuous 5 times, then respectively at 7,9, It is immunized within 11 weeks three times, amounts to 8 times.Diabetic mice docking is taken into blood before immune every time, take when blood detected with blood sugar monitoring instrument it is small Mouse blood glucose level.The results are shown in Table 4, the experimental results showed that, D41-IP, D41-IPA, D41-IAP, D41-2-IP, D41-3- IP、D41-4-IP、D41-6-IP、O-IP、 O-IPA、O-IAP、O2-IP、O-2-IP、O-4-IP、U-IP、A1-IP、A2-IP、 It can be significant after PDL2-IP, IA2 (5)-O-P2-1, IA2 (5)-D41--P2-1, the immune diabetic mice of D41-I6P, O-I6P Property reduce mouse blood sugar, have statistical difference.It can develop for treating diabetes.

The blood glucose test results of the diabetic mice of peptide based immunogens immunization therapy STZ induction of the table 4 containing carrier peptides

P compared with IP ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05

Embodiment 4-7. is the peptide based immunogens D41-IP and D4-4-IP containing carrier peptides in immune protection NOD mouse 1 type sugar The hypoglycemic and death rate, disease incidence detect after urine disease model:

40 female NOD mices (are purchased from Fukang company, Beijing China, SCXK (capital) 2014-0004.) random point 4 groups, every group 10: solvent group, P277 group (positive controls), D41-IP group, D4-4-IP group.According to 7,19,20,21,22,23,25, 27,29 administrations.Four limbs oxter and dorsal sc multiple spot are immune.Administration group drug is 1mg/mL, 100 μ g/ of dosage only/time, Solvent group: 100mL finish Lipofundin/only/time.After peptide immunization therapy mouse, with docking blood taking method blood glucose meter results of regular determination The blood glucose of mouse.After NOD female mice is given containing carrier peptides peptide based immunogens, change of blood sugar, disease incidence and the death of mouse Rate situation is as shown in table 5.As the result is shown: peptide based immunogens D41-IP, D4-4-IP and control group compare, and can significantly control blood glucose, Morbidity and mortality, last mouse blood sugar restore normal, no morbidity and mortality.

The blood glucose of 5 peptide based immunogens immune protection NOD mouse of table, morbidity and mortality testing result

Embodiment 4-8. is the present invention peptide based immunogens D41-IP, D41-4-IP, O-IP, A1-IP, A2-IP containing carrier peptides The serum generated after the diabetes C57BL/6J hero mouse model of immunization therapy STZ induction inhibits DPP4 enzymatic activity, xanthine oxidation Enzymatic activity, the inhibiting rate test experience of acetylcholine esterase active:

D41-IP, D41-4-IP immune serum inhibit the inhibiting rate test experience of DPP4 enzymatic activity, experimental principle are as follows: hair Color substrate method.DPP4 hydrolyzes Gly-Pro-pNA, generates pNA (yellow), and pNA has characteristic absorption peak at 405nm, passes through enzyme Mark instrument measures absorbance value at 405nm, and carrying out the active height of reaction enzymes, (i.e. each group Serum Antibody is to DPP4 inhibition level Size).Specific experiment step are as follows: 1. are incubated for serum, buffer, enzyme 30 minutes respectively under 37 degrees Celsius；2. according to 96 orifice plates are added in the sequence of enzyme, buffer, substrate.Overall reaction system is 100uL, is divided into the blank control group (bottom 0.26mmol/L Object 5uL；Tris-HCl buffer 95uL), negative control group (0.5U/L DPP4 10uL；0.26mmol/L substrate 5uL； Tris-HCl buffer 85uL), positive controls (0.5U/L DPP4 10uL；0.26mmol/L substrate 5uL；5 times diluted Each group serum 10uL；Tris-HCl buffer 75uL), positive blank group (0.26mmol/L substrate 5uL；5 times of diluted each groups Serum 10uL；Tris-HCl buffer 85uL).3.37 DEG C of constant incubators react 60min, measure the absorbance at 405nm. 4. comparing the value of each group OD405, or calculate inhibiting rate.

The calculation formula of inhibiting rate is as follows: inhibiting rate (%)=(OD negative control-OD blank control)-(OD inhibitor-OD suppression Formulation blank control)/(OD negative control-OD blank control) x100%.

O-IP, O2-IP immune serum inhibit the oxidation of xanthine used in the inhibiting rate test experience of xanthine oxidase activity Enzyme is purchased from the graceful biotech firm of Hypon (Roche import packing).Reaction system are as follows:

By shown in table be loaded, be maintained at 25 DEG C and reacted, enzyme solution with after serum mixing 20min addition reaction system after It mixes, is poured into cuvette immediately, light absorption value is measured under 294nm wavelength, reaction 1min records OD value.Xanthine oxidase The calculating of inhibiting rate is according to the definition of activity of enzyme reaction, and under inhibitor existence condition, activity of enzyme reaction is expressed as ACT_i, Activity of enzyme reaction is expressed as ACTo in the presence of no inhibitor, then serum can determine the relative inhibition (I) of xanthine oxidase Justice are as follows:

The inhibiting rate test experience of serum acetylcholine esterase inhibition activity takes 96 hole microwell plates, is loaded by following grouping Screening.Sample well: AChE 20 μ L, ASCh (75mM) 60 μ L, DTNB (10mM) 80 μ L, 10 μ of compound to be screened or positive drug The 30 μ L of phosphate buffer (PBS) of L, 0.05molL-1；Enzyme control wells: 20 80 100 μ of μ L, PBS of μ L, DTNB of AChE L；ASCh control wells: 60 60 μ L of μ L, DTNB80 μ L, PBS of ASCh；Blank control wells: 80 μ L, PBS120 μ L of DTNB.Oscillation Uniformly, proper temperature is incubated for after a certain period of time, measures absorbance (A) at 412nm.A1-IP, A2-IP immune serum are to acetyl The inhibiting rate calculation formula of cholinesterase are as follows: inhibiting rate %=(A standard-A sample)/(A standard-A blank-A enzyme control-ASCh Control) × 100%.Enzyme inhibition rate experimental result is as shown in table 6, as the result is shown D41-IP, D41-4-IP, O-IP, A2-IP group It can inhibit serum DPP4 active significantly, there is significant difference compared with solvent group.

The inhibiting rate testing result of 6 peptide based immunogens immune serum inhibitory enzyme activity of table

P compared with IP ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05

Peptide based immunogens D41-IP, D41-4-IP, O-IP, O2-IP, U-IP difference of the embodiment 4-9. containing carrier peptides of the present invention The inspection of serum uric acid, creatinine and anti-oxidation stress index after the diabetes C57BL/6J hero mouse model of immunization therapy STZ induction It surveys；

The detection reagent of mice serum uric acid, creatinine and anti-oxidation stress is provided by building up company, is illustrated according to kit It is detected and is calculated.Experimental result is as shown in Table 7 and 8, the results showed that D41-IP, D41-4-IP, O-IP, U-IP are immunized small After mouse, serum creatinine and uric acid and level are remarkably decreased, and have significant difference compared with solvent group and IP.Antioxidation It significantly increases, there is statistical difference.Therefore diabetic complication can be alleviated, protect kidney.

The testing result of serum uric acid, creatinine after the immune STZ diabetic mice of 7. peptide based immunogens of table

P compared with IP ^*P < 0.05,^**P < 0.01；^#P < 0.05,^##P < 0.01；

8. peptide based immunogens of table be immunized STZ diabetic mice after serum oxidative stress index testing result

P compared with IA(5)-P2-1 ^**P < 0.01^*0.05@P < 0.05 of P <^#P < 0.05^&P < 0.05

It is small that Balb/c is immunized in embodiment 4-10. peptide based immunogens D41-IP, O-IP, U-IP containing carrier peptides of the present invention respectively Mouse prepares the application of monoclonal antibody by monoclonal antibody hybridoma technology::

Blbc/c mouse is immunized respectively with D41-IP or O-IP or U-IP, after immunization method is shown in embodiment 4-2-2. 2 weeks Potency (detection method is shown in embodiment 4-3.) is detected respectively up to 20000 (D41-I), 8000 (O-IP), 12000 (U-IP).Then Monoclonal antibody is prepared according to conventional hybridoma technology.

Hybridoma technology prepares monoclonal antibody, cell fusion process: (1) Sp2/0 cell in good condition is chosen, from Revolving speed 1000rpm time 5min is arranged in scheming, is centrifuged, and counts, and after cannoing be used up full culture medium washing, takes required cell number standby With.(2) after BALB/c mouse is put to death, splenocyte suspension is prepared, appropriate number of splenocyte is chosen according to Sp2/0 cell density. (3) Sp2/0 cell and splenocyte are mixed in 1: 5 ratio, is added into 50mL centrifuge tube, 1200rpm centrifugation.It (4) will training Feeding base is outwelled.(5) it is gently shaken with hand at centrifugation bottom of the tube cell precipitation, cell precipitation is made slightly to become loose.(6) 1mL is added The PEG solution of 37 DEG C of preheatings, uniform rotation centrifuge tube when dropwise addition.The culture medium of preheating is added in 90s, terminates fusion. (7) centrifuge speed 1000rpm time 6min, centrifugation are set.(8) retain cell precipitation, gently weighed with appropriate HAT culture medium It is outstanding, it cannot firmly be blown and beaten with liquid-transfering gun, prevent the cell of fusion from scattering.(9) according to feeder cells bed board the case where, HAT is added Culture medium reaches required volume.(10) bed board, 100 holes μ L/, stationary culture in incubator.(11) the 4th day after merging, HAT half, which is measured, changes liquid, and HT half is measured and changed liquid within the 8th day, and the 12nd day HT full dose changes liquid, and the 16th day with complete DMEM culture medium culture.

Filtering hybridoma and clone, the positive colony hole that the above method is screened use limiting dilution assay, carry out 3 subclones, obtain the hybridoma cell strain that can secrete the B cell epitope monoclonal antibody of anti-XOD.Limiting dilution assay is as follows: (1) using The same method in front prepares feeder cells suspension and bed board；(2) hybridoma to grow fine is chosen, is counted, control is thin Born of the same parents' number range is 1-5 × 10³/mL；(3) cell suspension is diluted to 10/mL with complete medium；(4) every 100 μ L cell of hole Suspension is added in 96 well culture plates；(4) 4-5d, after there is cell clone, supplemented medium are cultivated；(5) when cultivating 8-9d, have Cell conditioned medium antibody test is carried out when apparent cell clone, repeats detection 1-2 times；(6) positive of selection monoclonal growth Hole is transferred in 24 orifice plates and expands culture.

The collection of antibody ascites.8-10 week old BALB/c mouse is pre-processed, atoleine, 500 μ are injected intraperitoneally L/ is only；Inject hybridoma after 7 days, 5 × 10⁶A/only；Mouse peritoneal expansion status is observed at 7-10 days, acquires ascites, It can be acquired again after 2-3 days.Last time, which extracts ascites, can take and will extract ascites after the execution of mouse cervical dislocation, so Peritonaeum, which is provoked, with syringe needle afterwards extracts ascites again to prevent abdominal viscera from blocking pin hole.Ascites is acquired, revolving speed is arranged in centrifuge 5000rpm time 15min, centrifugation collect supernatant ascites and in -70 DEG C of freezen protectives, and spare, a mouse collects 2-3 times Ascites.

Antibody preparation and purification process (1) octanoic acid-ammonium sulfate precipitation method preliminary purification, first low-speed centrifugal is by contaminating cell It removes, then high speed centrifugation removes smudge cells residue etc., crosses 0.22 μm of filter membrane and remove fibrin clot etc..Ascites is gone Except antibody is tentatively extracted with octanoic acid-ammonium sulfate precipitation method after impurity, ELISA detects antibody titer.

Embodiment 4-11.ELISA detection peptide based immunogens D41-IP, O-IP, U-IP pass through after Balb/c mouse is immunized respectively The potency of the monoclonal antibody of monoclonal antibody hybridoma technology preparation:

The titer detection method of monoclonal antibody antibody is same as above, as a result as shown in Table 9: Balb/ is immunized in immunogene D41-IP The potency of the monoclonal antibody CPU-KD4 prepared after c mouse by monoclonal antibody hybridoma technology is immune for 32000, O-IP The potency of the monoclonal antibody CPU-K01 prepared after Balb/c mouse by monoclonal antibody hybridoma technology is 32000, U- The potency of monoclonal antibody CPU-KU1 prepared after Balb/c mouse by monoclonal antibody hybridoma technology is immunized in IP 16000-32000。

The bioactivity result of the monoclonal antibody prepared after mouse is immunized in peptide based immunogens of the table 9. containing carrier peptides

Western Blot experiment of the embodiment 4-12. monoclonal antibody in conjunction with antigen-specific:

The hybridoma of the energy secretory antibody of Balb/c mouse preparation is immunized in immunogene D41-IP, O-IP, U-IP, protects There are Wuhan University, strain number is shown in Table 10..The specificity of detection antibody is tested with Western Blot, detection method is shown in embodiment 4-4.As a result see that Fig. 2 is shown: the monoclonal antibody CPU-KD4 prepared with a hybridoma can be with people DPP4 specific bond (figure 2A), the monoclonal antibody CPU-K01 energy and xanthine oxidase specific bond (Fig. 2 B) prepared with a hybridoma, with one The monoclonal antibody CPU-KU1 of hybridoma preparation can be with 1 specific bond of urate transporter (Fig. 2 C).Antibody can be used In the detection reagent of exploitation detection related antigen.

The test experience of embodiment 4-13. monoclonal antibody hypotype:

Mei Mian company kit is bought, detection method is the same as embodiment 4-5.Testing result is shown in Table 10, and display antibody is IgG1, IgG1 are Th2 antibody in mouse.

The hypotype testing result of the monoclonal antibody prepared after mouse is immunized in peptide based immunogens of the table 10. containing carrier peptides

Embodiment 4-14.D41-IP and the hypoglycemic experiment of monoclonal antibody CPU-KD4:

With STZ induction C57BL/6J hero mouse diabetes model (method is shown in embodiment 4-1) and NOD mouse type 1 diabetes mould Type (15 week old), 500ug/ mouse of CPU-KD4 intravenously administrable and D41-IP administration 100ug/, weekly intravenously administrable 1 time. Solvent group is PBS buffer solution, detects blood glucose with blood sugar test paper.

STZ mouse fasting blood glucose and NOD mouse non-fasting blood sugar monitoring result are as shown in Table 11: 500ug/ is only administered CPU-KD4 can significantly reduce blood glucose, there is extremely significant difference, statistically significant (^{* *,}P < 0.001).Illustrate that this monoclonal is anti- Body can be used for treating diabetes.

The monoclonal antibody CPU-KD4 treatment STZ induction that table 11. is prepared after peptide based immunogens and its immune mouse containing carrier peptides Diabetic mice and NOD mouse type 1 diabetes model blood glucose test results

P compared with IA(5)-P2-1 ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05

The experiment of embodiment 4-15. monoclonal antibody anti-trioxypurine:

Subcutaneously induce 6 week old Kunming mouse high lithemia models, monoclonal antibody CPU- weekly with 250mg/kg Oteracil Potassium 300ug/ mouse of K01, CPU-KU1 intravenously administrable, weekly intravenously administrable 1 time.Solvent group is PBS buffer solution, uses Nanjing weekly It builds up and uric acid reagent box detection uric acid is provided.

Testing uric acid result is as shown in Table 12: CPU-K01, CPU-KU1 is only administered in 300ug/, can significantly reduce uric acid, has Significant difference, statistically significant (^*,P < 0.01).Illustrate that this monoclonal antibody CPU-KO1 and CPU-KU1 can be used for controlling Treat hyperuricemia.

The high urine of the mab treatment Oteracil Potassium induction prepared after mouse is immunized in peptide based immunogens of the table 12. containing carrier peptides The uric acid testing result of sour mouse

P compared with IA(5)-P2-1 ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05

Embodiment 4-16. monoclonal antibody CPU-KD4 and CPU-K01 inhibit DPP4 enzymatic activity and xanthine to aoxidize respectively The inhibiting rate of enzymatic activity tests (method is shown in embodiment 4-8):

Enzyme inhibition rate experimental result is as shown in table 13, and monoclonal antibody CPU-KD4 and CPU-K01 group can divide as the result is shown Do not inhibit people DPP4 and xanthine oxidase activity significantly, there is significant difference compared with solvent group.

The inhibiting rate inspection of the monoclonal antibody inhibitory enzyme activity prepared after mouse is immunized in the single peptide based immunogens containing carrier peptides of table 13. Survey result

P compared with IP ^{* *,}P < 0.001；^*,P < 0.01；^*P < 0.05

Blbc/c mouse or immune five feature mouse 2 times is immunized with peptide based immunogens such as D41-IP or O-IP or U-IP, finally Potency is detected after primary immunization 2 weeks in 8000-20000, is taken spleen cell, is extracted genome or total serum IgE, it is anti-according to monoclonal Body technique or phage antibody library technique method or other methods for needing immunogen immune to prepare antibody, can prepare various Monoclonal antibody or genetic engineering antibody include Humanized single chain antibody, Fab antibody or macromolecular antibody etc..Company is sent to measure The variable region sequences of above-mentioned antibody are analyzed with related software and obtain 3 pairs of CDR regions, and it is anti-can to prepare small molecule on this basis Body, for preventing and treating disease relevant to peptide epitopes and complication and exploitation detection reagent.

Conclusion: carrier peptides of the present invention and target antigen albumen such as people DPP-4 or xanthine oxidase or urate transporter 1 or the B cell antigen epi-position or haptens peptide of human acetylcholinesteraseisomer connect to induce with different carriers and specifically target mesh Mark antigen protein and its B cell epitope or haptens peptide Th2 or Th1 antibody (in Mice Body Th1 can generate IgG2a and IgG2b subclass antibodies, Th2 type can then generate IgG1 subclass antibodies, and Th1 can generate IgG1 subclass antibodies, Th2 type in mankind's body IgG2a hypotype can then be generated.When B cell antigen epi-position or haptens peptide are connected with common carrier BSA or KLH of the invention, Immune biology generates high price specific antibody, can prepare antibody or Th1 and Th2 monoclonal antibody and all kinds of genetic engineering antibodies； When B cell antigen epi-position or haptens peptide are connected with carrier peptides of the invention, it is special anti-that immune biology can produce high price Body can prepare antibody or Th2 monoclonal antibody and all kinds of genetic engineering antibodies, and it is thin that Th2 subclass antibodies can increase Th2 class The generation of intracellular cytokine simultaneously controls the higher disease of Th1, alleviates symptom), and generate the energy and target antigen specificity knot of higher level The antibody of conjunction inhibits enzyme activity or GAP-associated protein GAP function, and blood glucose can be significantly reduced in peptide based immunogens, inhibits DPP-4 activity, base It also can significantly reduce blood glucose in the monoclonal antibody of peptide based immunogens preparation, such as containing the B cell antigen epi-position of DPP-4 or its transformation Potency reaches 20000 after animal is immunized in the peptide based immunogens of epitope peptide, can be used for preparing the specific antibody of anti-DPP-4, preparation Antibody also inhibits DPP-4 active, can significantly reduce blood glucose, can develop prevention and treatment and detection reagent；In addition, as contained xanthine oxidase Potency reaches 8000 after animal is immunized in the B cell antigen epi-position or its peptide based immunogens that epitope peptide is transformed for changing enzyme, inhibits xanthine Oxidase active, can be used for the specific antibody for preparing anti-yellowing purine oxidase, and the antibody of preparation also inhibits xanthine to aoxidize Enzymatic activity and it can significantly reduce uric acid；The specific antibody prepared on the basis of this peptide based immunogens can be combined with target antigen, can also To develop detection reagent use.B cell antigen epi-position or its peptide based immunogens that epitope peptide is transformed such as containing urate transporter 1 Potency reaches 12000 or so after immune animal, can be used for the special Th2 antibody for preparing anti-urate transporter 1, prepares Th2 antibody can significantly reduce uric acid and anti-oxidation stress.Treatment and detection reagent can be developed.When selection target antigen The prediction B cell epitope or synthetic peptide haptens of albumen human acetylcholinesteraseisomer or PD-L1 or neuraminidase, by itself and this Class carrier peptides connect to form peptide based immunogens A1-IP, A2-IP, PDL2-IP etc., and the effect of specificity can be also generated after immune mouse Valence is up to 14000-16000, and acetylcholine esterase inhibition activity can be developed and be used to prepare antibody especially Th2 antibody IgG1 (mouse) or human IgG2 a (people) is for treatment Th1/Th2 dysequilibrium related disease or detection.Examples detailed above explanation, this The carrier peptides of invention and the immunogene of the peptide containing examples of such carriers have important and extensive use with immunologic in medicine.

Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although passing through ginseng According to certain preferred embodiments of the invention, invention has been described, but those skilled in the art should manage Solution, can make various changes, without departing from defined by the appended claims to it in the form and details The spirit and scope of the present invention.

Claims (10)

1. the similar carrier peptides of a kind of composed structure, it is characterised in that: carrier peptides contain a polypeptide sequence, which contains There are amino acid sequence 1 and amino acid sequence 2, amino acid sequence 1 and amino acid sequence 2 pass through the connection of flexible peptide or joining peptide Such carrier peptides is formed, the amino acid sequence 1 is the B cell epitope IA2 (5) of insulinoma GAP-associated protein GAP IA-2, shown ammonia Base acid sequence group prejudice SEQ ID No.1, the amino acid sequence 2 are the Th2 cell epitope P2 of the C-terminal of P277 and its change structure Peptide, shown amino acid sequence group prejudice SEQ ID No.2.

2. it is according to claim 1 flexibility peptide or connection peptide linker be X1X2X3, --- --- Xn, --- --, X1X2X3X4X5, X1X2X3X4, X1X2X3, X1X2, X, GGGGG, GGGG, GGG, GGS, GG, G, K, KK or other small peptides. X1X2X3X4, X1X2X3X4X5, X1X2X3X4 X5 X6 and X1X2X3------Xn, the amino acid sequence are shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, Xn are any amino acid residue.The preferred 1-20 of n I Uncle's number.

3. carrier peptides according to claim 1 to 2, when the C-terminal of IA2 (5) and the N-terminal of P2 peptide fragment pass through flexible peptide or connection The connection of peptide fragment forms a kind of carrier peptides IP, IPA, IAP, 2-IP, 3-IP, 4-IP, 6-IP and I6P amino acid sequence is shown in respectively SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、 SEQ ID NO.13 and SEQ ID NO.14, or the C-terminal of IA2 (5) peptide fragment FEYQD and B cell epitope or peptide haptens are passed through After flexible peptide or link peptide are covalently attached, then formed by the connection of flexibility peptide or joining peptide with the N-terminal of Th2 epitope P2 peptide fragment The amino acid sequence of peptide based immunogens IA2 (5)-B-P2-1 etc., IA2 (5)-D41-P2-1 and IA2 (5)-O-P2-1 are shown in SEQ respectively ID NO.15 and SEQ ID NO.16.

4. carrier peptides according to claim 1 to 3, when B be respectively people DPP4, people's xanthine oxidase, human urine acid transporter When the advantage B cell epitope or synthetic peptide haptens of albumen 1 and human acetylcholinesteraseisomer and carrier peptides by link peptide distinguish structure At peptide based immunogens D41-IP (amino acid sequence is shown in SEQ ID NO.17), (amino acid sequence is shown in SEQ ID to D41-2-IP NO.18), D41-3-IP (amino acid sequence is shown in SEQ ID NO.19), D41-4-IP (amino acid sequence is shown in SEQ ID NO.20), D41-6-IP (amino acid sequence is shown in SEQ ID NO.21) O-IP (amino acid sequence is shown in SEQ ID NO.22), O-2-IP (amino Acid sequence is shown in SEQ ID NO.23), O-4-IP (amino acid sequence is shown in SEQ ID NO.24), D41-IPA (be shown in by amino acid sequence SEQ ID NO.25), D41-IAP (amino acid sequence is shown in SEQ ID NO.26), (amino acid sequence is shown in SEQ ID to O-IPA NO.27), O-IAP (amino acid sequence is shown in SEQ ID NO.28), D41-I6P (amino acid sequence is shown in SEQ ID NO.29), O- I6P (amino acid sequence is shown in SEQ ID NO.30).

5. the target antigen of energy described in -4 is connected with carrier peptides according to claim 1 epitope containing B cell or peptide haptens, packet Include but be not limited to the relevant target antigen of the higher disease of Th1.

6. carrier peptides described in -5 and peptide based immunogens and derivative and its coupling protein and fusion protein can also according to claim 1 To make them as production plant, be able to produce thin containing this B for recombinant microorganism and transgenic animals or plant and its cell Born of the same parents' epitope and polypeptide immunogen and coupling protein antigen and its fusion protein containing this epitope or production oral vaccine or production DPP4 special Th2 antibody and its derivative and analogue, drug.

7. peptide based immunogens containing carrier peptides described in the claim 1-6 comprising immunological effective amount and its coupling protein and melting The composition of hop protein or series connection object, it is characterised in that: also comprising pharmaceutically acceptable carrier and pharmaceutically acceptable auxiliary Material.Changing the Antigenic Peptide that structure obtains comprising this polypeptide and polypeptide immunogen and its coupling protein and fusion protein and thus Antigenic Peptide is The preparation of effective component, polypeptide and immunogene are to have with coupling protein or fusion protein that pharmaceutically acceptable carrier is connect Imitate injection, sustained release agent, subdermal implants, tablet, pulvis, granule, capsule or the oral solution of the preparation of ingredient.

8. according to claim 1 the peptide based immunogens described in -7 containing carrier peptides and thus polypeptide change immunogene that structure obtains and its Immunogenic composition etc. is used to enhance the immunogenicity of B cell epitope or peptide haptens, this antibody is Th2 hypotype, and feature exists In: inhibit enzyme activity or GAP-associated protein GAP function, can be used for preparing vaccine antibody for prevention and treatment Th1 is higher or relevant to peptide epitopes disease And complication.

9. the peptide based immunogens described in -7 containing carrier peptides can induce high price specifically to target target antigen egg according to claim 1 (Th2 type can generate IgG1 subclass antibodies to the Th2 antibody of its B cell epitope of bletilla or haptens peptide in Mice Body, in mankind's body Th2 type can then generate IgG2a hypotype), antibody or Th2 monoclonal antibody and all kinds of genetic engineering antibodies, Th2 hypotype can be prepared Antibody can inhibit enzyme activity or GAP-associated protein GAP function, increase the generation of Th2 type cytokines and control the higher disease of Th1, alleviate Symptom), and generate higher level energy and target antigen specific binding antibody, peptide based immunogens and based on peptide based immunogens prepare Monoclonal antibody can significantly reduce blood glucose or reduce uric acid, inhibit enzyme activity or GAP-associated protein GAP function.

10. the immunogene described in -9 containing carrier peptides and the antibody thus produced and derivative and modification according to claim 1, It can be used for preparing medical treatment target antigen exception, pathoglycemia, oxidative stress abnormal, dysglycemia and Th1 be higher draws The diabetes and its complication risen, nephrosis, hyperlipidemia, hyperuricemia, atherosclerosis, rheumatic arthritis, old age are silly The prevention and treatment of the related diseases such as slow-witted, eye disease, pedopathy, angiocarpy, and for detecting related specific target antigen, exploitation detection Reagent.