CN108586619A - It is a kind of to prevent and treat type 1 diabetes and with linear 33 peptide and application thereof of protection Pancreatic beta cells function - Google Patents

It is a kind of to prevent and treat type 1 diabetes and with linear 33 peptide and application thereof of protection Pancreatic beta cells function Download PDF

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CN108586619A
CN108586619A CN201810434955.6A CN201810434955A CN108586619A CN 108586619 A CN108586619 A CN 108586619A CN 201810434955 A CN201810434955 A CN 201810434955A CN 108586619 A CN108586619 A CN 108586619A
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peptide
diabetes
cell
dpp4
amino acid
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李泰明
顾小骞
焦瑞
李志鑫
方金芝
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China Pharmaceutical University
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China Pharmaceutical University
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Abstract

The invention discloses linear 33 peptide molecules,It is made of a B epitope (10 peptide) for people's dipeptidyl peptidase 4 and 23 peptides of an epitope containing Th2,The antibody of energy and DPP4 specific binding of the serum containing high price after animal is immunized in 33 peptides,Antibody can inhibit DPP4 activity and adjust the immune rebalancing for tending to Th2,It is prevented with 33 peptide subcutaneous inoculations every 2 weeks or is treated type 1 diabetes NOD mouse and treat the diabetic mice of STZ inductions,Significantly control or reverse blood glucose,NOD mouse blood sugars are normal,GLP 1 and Th2 cell factors significantly increase,Glucagon and Th1 cell factors significantly reduce,Significantly improve incidence,The death rate,Panimmunity balances and uric acid,Creatinine and blood fat and oxidative stress index,Pancreas inflammatory mitigates,Pancreatic secretion insulin ability significantly increases.33 peptides and include the vaccine and drug prepared with microorganism, animal and plant and cell expression based on 33 peptides, have significant application value preventing and treating in the diseases such as diabetes especially type 1 diabetes and complication.

Description

It is a kind of to prevent and treat type 1 diabetes and with the line of protection Pancreatic beta cells function 33 peptides of property and application thereof
Technical field
The present invention relates to immune, pharmacy and medicine related fields, and type 1 diabetes can be prevented and treat and have by being related to one Protect linear 33 peptide of Pancreatic beta cells function and its in purposes medical and being immunized.This 33 peptide is by people's dipeptidyl peptidase 4 23 peptides of the B cell epitope of one 10 peptide of (Dipeptidyl peptidase 4, DPP4) and an epitope containing Th2 composition, Serum after the immune animal of this 33 peptide contains the high-caliber antibody that can be specifically bound with DPP4, this antibody can inhibit DPP4 to live Property and adjust tend to Th2 immune rebalancing, every 2 weeks use this 33 peptide subcutaneous administration, prevention or treatment generally acknowledge type 1 diabetes The diabetic mice of NOD mouse and treatment STZ inductions, significantly can control or reverse blood glucose, GLP-1 and Th2 cell factors notable It increases, pancreas glucagon and Th1 cell factors significantly reduce, and uric acid, creatinine and blood fat and oxidative stress index significantly change Become, normal blood glucose, the blood glucose in diabetic mice of STZ inductions are shown by the type 1 diabetes NOD mouse for preventing or treating It significantly improves, is less than diabetes glucose morbidity index, while pancreas is protected, pancreas inflammatory mitigates, pancreatic secretion insulin Ability significantly increases.This 33 peptide is preparing the relevant diseases such as prevention and treatment diabetes especially type 1 diabetes and its complication There is significant application value on drug.
Background technology
Diabetes (Diabetes mellitus, DM) are the metabolic diseases characterized by insulin relative or absolute deficiency Disease, it is type 1 diabetes that insulin, which absolutely lacks, and the opposite shortage of insulin is diabetes B.Type 1 diabetes also known as insulin according to It is a kind of pancreas specific Damage that Th1 is cell-mediated to rely patients with type Ⅰ DM (Type 1 diabetes mellitus, T1DM) Autoimmune disease.The insulin secretory cell of patient's body --- beta Cell of islet makes it by the immune system attack of itself It goes to pot, afunction, caused diabetes is absolutely lacked so as to cause insulin.Insulin is generated since β cells have With the function of adjusting blood glucose, the breaking-up of β cell masses has eventually led to the imbalance of blood glucose and the generation of hyperglycemia.
Dipeptidyl peptidase 4 is otherwise known as t cell surface antigen CD26, is a kind of serine protease of cell surface.People Earliest find DPP4 can be used as one treatment diabetes B important target spot.Exactly because it is to other intestines such as GLP-1, GIP The degradation of pancreotropic hormone.There is GLP-1 the insulin secretion accelerating of glucose dependency, glucagon suppression secretion to promote β thin Born of the same parents regenerate and repair, and delay postprandial gastric emptying, and increasing functions, the GIP such as satiety equally has the function of insulin secretion accelerating.So And the half-life period in vivo such as GLP-1, GIP only has 1-2min, degraded by DPP4 rapidly inactivates later.DPP4 inhibitor passes through competing The active site of striving property combination DPP4, reduces the catalytic activity of enzyme, and the level to increase internal GLP-1 and GIP reaches promotion The effect of insulin secretion, stability contorting blood glucose improves β cell functions, and patient's weight will not be caused to increase, and can avoid Risk of hypoglycemia.The chemical synthetic drugs such as current a variety of DPP4 inhibitor such as Xi Gelieting, saxagliptin, are widely used to clinic Although also having been reported that treatment of the DPP4 inhibitor for type 1 diabetes (Type 1 diabetes mellitus, T1DM) at present, But since it is chemicals, be frequently accompanied by long-term use side effect, especially suffer from secretion metabolic disease with hypertension, The middle-aged and the old of hyperlipidemia and hyperglycemia, control bad leads to compromised kidneys for a long time.Therefore it needs to develop new DPP4 inhibition Agent and the immunomodulator that can also adjust Th1 trends Th2 simultaneously.
In the diseases such as rheumatoid arthritis, type 1 diabetes, multiple sclerosis and chronic thyroiditis, Th1 or Th1 The cell factor of secretion is dominant, and Th1 induction morbidities aggravate the state of an illness；It can then mitigate illness to Th2 drifts, or even prevent morbidity, This is related with the inflammatory of Th1/Th2 and anti-inflammatory response.Relationship based on Th1/Th2 drifting states Yu various diseases, increasingly More research is just tended to find, develops the drug and method that can reverse or stablize Th1/Th2 states.Th1 can be produced in Mice Body Raw IgG2a subclass antibodies, Th2 types then will produce IgG1 subclass antibodies, but be opposite in human body.Th2 subclass antibodies can To increase the generation of Th2 type cytokines and control the higher diseases of Th1, treatment and alleviation disease symptoms.The specificity of antibody It is to be directed to epitope, B cell epitope (B cell epitope) is antigen molecule by antibody or B cell antigen receptor (B cell Receptor, BCR) specific recognition and the special chemical group that is combined with each other, also known as antigenic determinant (antigenic Determinant, AD).The determination of B cell epitope to the synthesis of polypeptide vaccine, the preparation of diagnostic reagent, monoclonal antibody sieve The research work such as choosing are of great significance.B cell epitope is divided into continuous type and discrete.B cell Antigen Epitope Prediction software： DNAStar, PREDITOP, ADEPT, PEOPLEOMIGA, UWGCG, ANTHEPROT etc..Hypotype and the B epitope coupling of antibody In relation to related, the peptide and albumen that screening is suitably connected with B epitope are difficult for carrier and immune adjuvant types, but to treatment Property vaccine research success it is significant.
Invention content
Type 1 diabetes can be prevented and treated the object of the present invention is to provide one and with protection Pancreatic beta cells function Linear 33 peptide and its medicine and it is immune on purposes.This 33 peptide is named as CPU-PDLP1.
In view of this, one of the objects of the present invention is to provide people's dipeptidyl peptidases 4 of B cell Antigen Epitope Prediction software prediction The amino acid sequence for the B cell epitope that one of (Dipeptidyl peptidase 4, DPP4) is made of 10 amino acid residues Row, are named as D1, shown amino acid sequence is：Val Phe Leu Glu Asn Ser Thr Phe Asp Glu (single-letter sequences Row：VFLENSTFDE), the amino acid sequence is shown in SEQ ID No.1.It connects and passes through with 23 peptides of an epitope containing Th2 It is 10 peptides for having high immunogenicity to be subcutaneously injected and animal proof D1 is immunized, and can generate the specific antibody of high titre in vivo, This antibody can be with people's DPP4 specific bindings.
The second object of the present invention is to provide the 23 of the connection of Peptide D 1 of an energy and above-mentioned 10 amino acid residues composition The amino acid sequence of peptide, this 23 peptide are named as LP1, and shown amino acid sequence is：GFEYQDSGTIIPALDSLTPANED, it is described Amino acid sequence is shown in SEQ ID No.2.For LP1 from 1 Th2 cell epitopes transformation, this Th2 cell epitope derives from people Re Xiu 1 and the relevant P277 peptides of diabetes of gram upper 437~460 of albumen HSP60, the N-terminals of P277 peptides have 1 by 13 amino acid The Th2 epitope P2 of composition form LP1 as soon as adding section connection peptide being made of 10 amino acid residues in its N-terminal.10 ammonia The amino acid sequence of connection peptide LP1 of base acid residue composition is：GFEYQDSGTI, the amino acid sequence are shown in SEQ ID No.3, It is named as L1, wherein deriving from human pancreatic island cell specific antigen one insulinoma GAP-associated protein GAP -2 (IA-2) membrane-proximal region containing one A B cell epitope of JM2, amino acid sequence are：FEYQD is named as IA2 (5).
The third object of the present invention is to provide the composition and amino acid sequence of 33 Peptide C PU-PDLP1.Shown in CPU-PDLP1 Amino acid sequence is：VFLENSTFDEGFEYQDSGTIIPALDSLTPANED, the amino acid sequence are shown in SEQ ID No.4.Its It is made of the C-terminal of a B cell epitope D1 and the N-terminal of above-mentioned 23 peptide LP1 of above-mentioned people DPP4 by being covalently keyed.
The fourth object of the present invention is to provide effect and the purposes of 33 Peptide C PU-PDLP1.A B cell epitope of DPP4 D1 is connected with the 23 peptide LP1 containing 1 Th2 cell epitope, and 23 peptides can stimulate and enhance B cell epitope D1 to generate in vivo specifically Property antibody ability, the antibody that animal can generate the energy and DPP4 specific bindings of high titre is immunized in 33 peptides, this antibody can inhibit DPP4 activity and adjust tend to Th2 immune rebalancing, per 2-3 weeks with this 33 peptide subcutaneously or intravenously administrable, can prevent or control The diabetic mice for treating generally acknowledged type 1 diabetes NOD mouse and treatment STZ inductions, significantly can control or reverse blood glucose, GLP-1, Insulin and Th2 cell factors significantly increase, and pancreas glucagon and Th1 cell factors significantly reduce, by preventing or controlling The type 1 diabetes NOD mouse for the treatment of show that normal blood glucose, the blood glucose in diabetic mice of STZ inductions significantly reduce, diabetes Complication index is such as：Uric acid, creatinine and blood fat and oxidative stress index significantly improve, while pancreas is protected, pancreas inflammatory Mitigate, pancreatic secretion insulin ability significantly increases.This 33 peptide prepare prevention and treatment diabetes especially type 1 diabetes and Protecting pancreas function and alleviating has significant application value on the drugs of relevant diseases such as diabetic complication.
The fifth object of the present invention is to provide：Recombinant microorganism is can be used for according to this 33 Peptide C of convention PU-PDLP1 With transgenic animals or plant and its cell, make them as production plant, can produce coupling protein containing CPU-PDLP1 and Fusion protein makes oral or other types pharmaceutical vaccine and its conjugate and composition pharmaceutical preparation.
The sixth object of the present invention is to provide the combination comprising 33 Peptide C PU-PDLP1 are contained described in immunological effective amount Object, coupling protein and fusion protein or connection in series-parallel object or arborescence.
The seventh object of the present invention is to provide the composition, coupling protein, fusion protein or connection in series-parallel object or branch Shape object is pharmacy and biologically conventional acceptable substance and structure, can be used for preparing vaccine and pharmaceutical preparation, be used for Prevent and/or treatment diabetes include 1 type and diabetes B and its complication and the raised relevant diseases of Th1, including is immune flat Weigh exception, oxidative stress exception, uric acid creatinine exception, nephrosis, dyslipidemia, atherosclerosis, senile dementia, eye disease, foot Application in the diseases such as disease, angiocarpy.
Further, also include pharmaceutically acceptable carrier or pharmaceutically acceptable auxiliary material.
Further, pharmaceutically acceptable carrier, including but not limited to：Heat shock protein HSP60/65, HSP70, Men Dong Amidase, 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, including but not limited to：Filler, diluent, excipient, adjuvant Deng.Such as include but not limited to：Cell factor, lactose, sucrose, glucose, starch, cellulose family (such as carboxymethyl cellulose, hydroxyl Third methylcellulose etc.), ethylene glycol, soya-bean oil, sesame oil, ethyl alcohol, sterile saline, sterile water, mannitol adds Lipofundin etc..
The beneficial effects of the present invention are：
The invention discloses the complete amino acid sequences of a 33 Peptide C PU-PDLP1, and ingredient is single, simple in structure, pendulum The influence of the bad epitope in natural DPP4 is taken off, by the stimulation of Th2 epitopes, immune response is with strong points, and antibody specificity is high, It can inhibit DPP4 activity and adjust the immune rebalancing for tending to Th2.The connection in series-parallel object of the PU-PDLP1 containing this 33 Peptide C, composition and Its coupling protein and fusion protein or branch object and its nucleic acid sequence can also directly be developed into prevention and treatment vaccine uses, Vaccine mainly prevents and treats but be not restricted to that prevent and treat include DPP4 exceptions, pathoglycemia, Th1/Th2 is unbalance, CD4 +/CD8+ is abnormal, Th17 and Treg are abnormal, diabetes caused by dysglycemia include 1 type and diabetes B and its concurrent Disease includes nephrosis, atherosclerosis, uric acid creatinine blood fat oxidative stress exception, senile dementia, eye disease, pedopathy, angiocarpy etc. The application of disease higher Th1.With most important theories value and broad prospect of application, there is important social and economic benefit.
Description of the drawings
In order to make the object of the invention, technical solution and advantage understand, the present invention is further retouched in detail in conjunction with attached drawing It states, wherein：
Serum after the diabetes C57BL/6J hero mouse that Fig. 1 induces for the immune STZ of 33 Peptide C PU-PDLP1 of the present invention and people The Western Blot experimental results of DPP4 specific bonds, wherein swimming lane 1 are Protein Marker (kDa), swimming lane 2-5 difference For people's DPP4 enzymes, D1-BSA, BSA and unrelated protein asparaginase；
Fig. 2 is the 1 type glycosuria of diabetes C57BL/6J heros mouse and prevention that 33 Peptide C PU-PDLP1 of the present invention treats STZ inductions Pancreas HE inflammation grades and picture testing result after sick NOD mouse, (Fig. 2A is C57BL/6J hero mouse pancreas HE inflammation grade figures, Fig. 2 B are NOD mice pancreatics HE figures)；
Fig. 3 is that pancreatic insulin is exempted from after 33 Peptide C PU-PDLP1 of the present invention treats the diabetes C57BL/6J hero mouse that STZ is induced Epidemic disease histochemical analysis testing result；
Fig. 4 is that 33 Peptide C PU-PDLP1 of the present invention prevents pancreatic insulin and glucagon after type 1 diabetes NOD mouse Immunohistochemical analysis testing result, (Fig. 4 A are pancreatic insulin assays figure, and Fig. 4 B are pancreas glucagon analysis chart)；
Fig. 5 is that pancreas CD4+T and CD8+T cell is exempted from after 33 Peptide C PU-PDLP1 of the present invention prevents type 1 diabetes NOD mouse The analysis result of epidemic disease group, CD4+T cells (Fig. 5 A), CD8+T cells (Fig. 5 B)；
Fig. 6 be 33 Peptide C PU-PDLP1 of the present invention prevent spleen cell flow cytometer detection Th1 after type 1 diabetes NOD mouse, (Fig. 6 A are Th1 to the analysis result of Th2, Th17 and Treg expression, and Fig. 6 B are Th2, and Fig. 6 C are Th17, and Fig. 6 D express for Treg；
Fig. 7 is that 33 Peptide C PU-PDLP1 of the present invention prevents glycosylated hemoglobin testing result after type 1 diabetes NOD mouse；
Fig. 8 is that 33 Peptide C PU-PDLP1 of the present invention prevents sugar tolerance experimental result after type 1 diabetes NOD mouse；
Specific implementation mode
Embodiment 1：The preparation and synthesis of the B cell D1 of people DPP4.
With B cell antigen epi-position analysis software and on-line analysis tool, in conjunction with secondary structure prediction and space structure Feature selects the advantage B cell antigen epi-position of several DPP4 from a epitopes up to a hundred, carries out animal immune screening, adaptive immune The B cell epitope D1 of the higher DPP4 of originality, amino acid sequence are shown in that SEQ ID NO.1, peptide are synthesized using FMOC solid-phase synthesis, HPLC detects purity ＞ 95%.
Embodiment 2：The preparation and synthesis of the 33 Peptide C PU-PDLP1 of the present invention of B cell antigen epi-position D1 containing the DPP4.
23 peptides that the Peptide D 1 of embodiment 2-1. structures and above-mentioned 10 amino acid residues composition connects, this 23 peptide are named as LP1, the amino acid sequence are shown in SEQ ID No.2.LP1 is from 1 Th2 cell epitopes transformation, this Th2 cell epitopes source In upper 437~460 1 P277 peptides of human heat shock protein HSP60, what the N-terminals of P277 peptides was made of by 1 13 amino acid Th2 epitope P2 form LP1 as soon as adding section connection peptide being made of 10 amino acid residues in its N-terminal.10 amino acid are residual The amino acid sequence of the connection peptide LP1 of base composition is shown in SEQ ID No.3, is named as L1, wherein thin from people's pancreas islet containing one A B cell epitope of born of the same parents' specific antigen-(IA-2) membrane-proximal region of insulinoma GAP-associated protein GAP -2 JM2, amino acid sequence are： FEYQD is named as IA2 (5).
Embodiment 2-2. by above-mentioned people DPP4 a B cell epitope D1 C-terminal and above-mentioned 23 peptide LP1 N-terminal By being covalently keyed 33 Peptide C PU-PDLP1 of composition, shown amino acid sequence is： VFLENSTFDEGFEYQDSGTIIPALDSLTPANED is shown in SEQ ID No.4.
The peptide or B cell antigen epi-position of above-mentioned narration are all made of the synthesis of FMOC solid-phase synthesis, and HPLC detects purity ＞ 90%.
Its specific building-up process is as follows：
1. selecting resin
For Peptide systhesis according to sequence inverse composition, polypeptide is connected since C-terminal is to N-terminal, Fmoc-Asp first (Otbu)- OH Wang Resin；
2. removing Fmoc
Reactor is added in deprotection liquid (20% hexahydropyridine and 80%DMF), 30min or so is blown with nitrogen, then takes out Fall, cleaned 5-6 times with DMF, then detect color of resin (generally in blue or brown).To remove before connecing next amino acid Blocking group on a upper amino amino.
3. the condensation of amino acid
Reactor is added in ready raw material and solid condensing agent (tbtu, hobt) after deprotection, DMF then is added simultaneously It is passed through nitrogen.Corresponding liquid activated dose (DIEA) is added after amino acid and condensing agent are completely dissolved to be reacted.Raw material with Solid activator inventory is typically all three times.After reacting the regular hour, a small amount of resin is taken to detect, when resin is in water white transparency When, show that the reaction was complete.
4. cutting
Synthetic polypeptide is added in cutting liquid, cutting liquid is filtered out after blender stirring 2-3h, is added wherein Ether, polypeptide can be precipitated.Polypeptide is obtained by filtration in filter paper, and then washing 4-5 times with ether obtains crude product peptide.
5. peptide purification
It is purified after crude product peptide is drained, obtains the sterling of HPLC purity ＞ 90%.
Experimental method in embodiment is unless otherwise instructed conventional method.Used kit is according to manufacture product Condition proposed by manufacturer carries out.
Embodiment 3：The diabetic rat model that STZ is induced is immunized in 33 Peptide C PU-PDLP1 of the present invention and type 1 diabetes NOD is small The immunogenicity and pharmacodynamic evaluation of mouse：
1, the foundation of diabetic rat model：
Streptozotocin (STZ) inducing mouse diabetes (DM) are injected intraperitoneally using low dose of continuous several times in embodiment 1-1. Model.
4 week old male C 57 BL/6 J mouses (are purchased from Yangzhou University's comparative medicine center, credit number：SCXR (Soviet Union) 2012- 0004.) 0.1M pH4.4 citrate buffers is used to configure a concentration of 7.5mg/ml of STZ, it is injected intraperitoneally by 50mg/kg dosage, One time a day, continuous injection 5 times measure blood glucose value in the 3rd, 7,14 day after modeling, with fasting blood-glucose twice in succession >= 11.1mmol/L is into mould standard.
NOD mouse type 1 diabetes models.
Female NOD mice, age of mouse 4~5 weeks, weight 18-20g are provided by Fukang bio tech ltd of Beijing China SCXK (capital) 2014-0004.
2, the prevention and treatment experiment of animal immune
40 STZ induced diabetes model mices of embodiment 2-1. are randomly divided into 4 groups, every group 10, respectively： Placebo groups (solvent control group, negative control group), P277 groups (positive controls), LP1 groups, CPU-PDLP1 groups.Administration group Drug concentration is 1mg/ml, and dosage is 100 μ g//times, i.e. 100 μ l liquids；Negative control group：100 μ l finishes/only/ It is secondary.Medicine ordinance method is dissolved in the ratio provisional configuration finish of 1ml20%Lipofundin in 1mg+40mg mannitol.In model Success (be subject to 8 hours blood glucose >=11.1mmol/L of fasting) is immunized on the 2nd week for the first time afterwards, weekly administration later 1 time, and continuous 5 It is secondary, then be immunized three times respectively at 7,9,11 weeks, amount to 8 times.
40 female NOD mices of embodiment 2-2. divide 4 groups, every group 10 at random：Solvent group (negative control group), P277 Group (positive controls), LP1 groups and CPU-PDLP1 groups.According to 7,19,20,21,22,23,25,27,29 administrations.Subcutaneous multiple spot Immune, four limbs oxter and back carry out prevention and are immunized.Administration group drug concentration is 1mg/mL, 100 μ of dosage g//times, the moon Property control group：100mL finishes Lipofundin/only/time.
30 female NOD mices of embodiment 2-3. divide 3 groups, every group 10 at random：Solvent group (negative control group), LP1 Group, CPU-PDLP1 groups.Start drug treatment when being higher than 13.9mmol/L according to postprandial blood sugar, after antibody generation, gives every 2 weeks 1 treatment of medicine.Subcutaneous multiple spot is immune, and four limbs oxter and back carry out prevention and be immunized.Administration group drug concentration is 1mg/mL, administration 100 μ of dosage g//times, negative control group：100mL finishes Lipofundin/only/time.
3, the diabetes C57BL/6J heros mouse model of 33 Peptide C PU-PDLP1 immunization therapies STZ inductions of the present invention and NOD mouse The immunogenicity and curative effect evaluation of type 1 diabetes model are tested：
Embodiment 3-1. is the diabetes C57BL/6J of 33 Peptide C PU-PDLP1 immunization therapies STZ inductions of invention The serum antibody ELISA and serum antibody titer testing result generated after male mouse model and NOD mouse type 1 diabetes models：
It is coated with dilution (0.05M sodium carbonate-bicarbonate buffer solutions, pH9.6) and dilutes D41-BSA, be made into final concentration of 0.1mL is added in 96 hole elisa Plates per hole for the solution of 0.5mg/mL, and 4 DEG C of coatings are overnight；Coating buffer is discarded, it is de- that 5% is added per hole Fat milk powder (pH7.4 PBS are prepared) confining liquid, 4 DEG C of full hole closings are overnight；Confining liquid is discarded, 1: 100 is added per hole, 5% degreasing The diluted Antigenic Peptide of milk powder immune mice serum 0.1mL, 37 DEG C of incubation 1h；Serum is discarded, (is spat containing 0.5% with PBST per hole The PBS solution of temperature -20) and distillation water spacer washing, each 3min, washing 6 times altogether；After washing, 5% skimmed milk power is added per hole The goat anti-mouse igg secondary antibody 0.1mL of diluted horseradish peroxidase label, 1: 5000,37 DEG C of incubation 1h of extension rate；Discard two Anti- liquid is washed per hole with PBST and distillation water spacer, and each 3min is washed 6 times altogether；After washing, it is aobvious that 0.1mL TMB are added per hole The H of 2mol/L is added after 37 DEG C are reacted 30min in color liquid2SO40.1mL terminates reaction；Distilled water returns to zero, microplate reader 450nm wavelength The lower OD values for measuring each hole.Experimental result is as shown in table 1, and mouse is after giving 33 Peptide C PU-PDLP1 of the present invention and being immunized, serum ELISA has pole significant difference the results show that generating the high-level specific antibody for DPP4 in serum with solvent and LP1 groups (P ＜ 0.001), potency reaches 20000.
1. 33 Peptide C PU-PDLP1 immune serum antibody ELISA testing results of table
P compared with IA(5)-P2-1 * *,P ＜ 0.001；*,P ＜ 0.01；*P ＜ 0.05；
Embodiment 3-2. is the diabetes C57BL/6J hero mouse moulds of 33 Peptide C PU-PDLP1 immunization therapies STZ inductions of the present invention The serum generated after type and NOD mouse type 1 diabetes models inhibits the inhibiting rate testing result of DPP4 enzymatic activitys：
It is Chromogenic assay that serum, which inhibits the inhibiting rate test experience principle of DPP4 enzymatic activitys,.DPP4 hydrolyzes Gly-Pro- PNA generates pNA (yellow), and pNA has characteristic absorption peak at 405nm, absorbance value is measured at 405nm by microplate reader, Carry out the active height of reaction enzymes (i.e. size of each group Serum Antibody to DPP4 inhibition levels).Specific experiment step is：1. will Serum, buffer solution, enzyme are incubated 30 minutes respectively under 37 degrees Celsius；2. according to enzyme, 96 holes are added in the sequence of buffer solution, substrate Plate.Overall reaction system is 100ul, is divided into blank control group (0.26mmol/L substrates 5ul；Tris-Hcl buffer solution 95ul), it is cloudy Property control group (0.5U/L DPP4 10ul；0.26mmol/L substrates 5ul；Tris-Hcl buffer solution 85ul), positive controls (0.5U/L DPP4 10ul；0.26mmol/L substrates 5ul；5 times of diluted each group serum 10ul；Tris-Hcl buffer solutions 75ul), positive blank group (0.26mmol/L substrates 5ul；5 times of diluted each group serum 10ul；Tris-Hcl buffer solution 85ul). 3.37 DEG C of constant incubators react 60min, measure the absorbance at 405nm.4. comparing the value of each group OD405, or calculates and inhibit Rate.
The calculation formula of inhibiting rate is as follows：(OD inhibitor-OD inhibits for inhibiting rate (%)=(OD negative control-OD blank controls)- Agent blank control)/(OD negative control-OD blank controls) x100%.Experimental result is as shown in table 2, as a result shows 33 Peptide C PU- PDLP1 can significantly inhibit serum DPP4 active, have significant difference compared with solvent group.
2. 33 Peptide C PU-PDLP1 immune serums of table inhibit the inhibiting rate testing result of DPP4 enzymatic activitys
Embodiment 3-3. is that serum after the diabetes C57BL/6J hero mouse of STZ inductions is immunized in 33 Peptide C PU-PDLP1 of the present invention It is tested with the Western Blot of people's DPP4 specific bonds：
Serum is carried out with people's DPP4 Western Blot experimental basis Western Blot standard practice instructions, by DPP4 It is transferred on pvdf membrane, jump condition 100V, 5h；Pvdf membrane after transfer as shaking at room temperature in 25mL Block buffers 1h.15mL TBST are washed 3 times (5min/ times)；Then pvdf membrane is soaked in 1: 10 diluted Antigenic Peptide immune serum, 1-2h or 4 DEG C of incubation at room temperature overnight, is slowly shaken, and 15mL TBST are washed 3 times (5min/ times).Add 1: 5000 diluted horseradish The sheep anti-mouse igg of peroxidase labelling, 37 DEG C of incubation 1h.PBST washes film 3 times, each 10min.DAB developing solutions are added to carry out Colour developing.Fully color development stopping in film transfer to deionized water is reacted after colour developing.DPP4 is after SDS-PAGE electrophoresis, and directly electricity turns Change onto pvdf membrane, after closing, for the serum obtained using 33 Peptide C PU-PDLP1 immunized mices as primary antibody, sheep anti-mouse igg is secondary antibody pair DPP4 carries out immuno absorbence, experimental result as shown in Figure 1, occur the purpose band that specific serum and DPP4 are combined on film, and It is not bound with other unrelated proteins, can be used for detecting special DPP4 antigen proteins.
Embodiment 3-4. is that the diabetes C57BL/6J heros mouse that STZ is induced is immunized in 33 Peptide C PU-PDLP1 of the present invention and NOD is small The detection of serum antibody hypotype after mouse type 1 diabetes model：
The assay kit of subtype-specific antibody is purchased from Mei Mian biotech firms, 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 per hole.2,100ul enzymes mark two is added per hole It is anti-, 37 DEG C of incubation 60min after preservative film winding.3, wash solution expire hole board-washing 5 times.4, per hole be added 50ul substrate As and 50ul substrate Bs, gently mixing, 37 DEG C are protected from light and are incubated 15min.5,50ul terminate liquids (2M H are added per hole2SO4) (color is become by indigo plant It is yellow).6, OD values are measured in 15min at 450nm wavelength.7, using standard items OD values as abscissa, concentration value is ordinate, is drawn Standard curve calculates sample concentration.Experimental result is as shown in table 3, the results showed that after the immune mouse of 33 Peptide C PU-PDLP1 in serum The specificity T h2 types IgG antibody 1 of generation, is significantly higher than LP1 and solvent group, has significant difference (P ＜ 0.01).IgG2b Significantly increase.Peptide epitopes and peptide based immunogens are immunized animal and can be used for preparing antibody.
The testing result of serum antibody hypotype after mouse is immunized in 3. 33 Peptide C PU-PDLP1 of table
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；0.01 , ＆＆P ＜ 0.01 of ＆P ＜
Embodiment 3-5. is the diabetes C57BL/6J hero mouse moulds of 33 Peptide C PU-PDLP1 immunization therapies STZ inductions of the present invention The hypoglycemic and death rate, incidence detect after type and NOD mouse type 1 diabetes models：
After peptide immunization therapy mouse, take the method for blood with the blood glucose of blood glucose meter results of regular determination mouse using docking.It is made through STZ After the C57BL/6J male mices and NOD female mices of mould give 33 Peptide C PU-PDLP1, the change of blood sugar situation such as table 4 of mouse It is shown.The experimental results showed that 33 Peptide C PU-PDLP1 be immunized after diabetic mice can conspicuousness and pole significantly reduce mouse blood sugar, There is significant difference (P ＜ 0.01, P ＜ 0.001) compared with solvent group and control group.Therefore above-mentioned peptide based immunogens can develop use In treatment diabetes with hypoglycemic.Statistics prevention type 1 diabetes NOD each groups mouse observed at 40 weeks during blood glucose, survival rate Change with incidence.The results are shown in Table 5, and the death rate of 33 Peptide C PU-PDLP1 is 0, (dead with negative control group (placebo) Die rate 35%) it compares, the survival rate of diabetic mice can be obviously increased.At the 21st week, 33 Peptide C PU-PDLP1 have 50% morbidity Rate, but be finally 0 in 40 weeks incidence, illustrate, midway is fallen ill, and can restore normal blood after the immunization therapy of peptide Sugar, and there is no hypoglycemia, it does not fall ill, prevention diabetes effect is notable；Statistics treatment type 1 diabetes NOD each group mouse are 40 Blood glucose, survival rate and incidence result of variations during week observation are shown in Table 6.At the 22nd week, 33 Peptide C PU-PDLP1 groups 100% Incidence, blood glucose are up to 27mmol/L, but finally from 30 weeks to 40 a week incidence be 0, and blood glucose is close to euglycemia, And there is no hypoglycemia, it does not fall ill, treatment diabetes effect is notable, can be used for preventing type 1 diabetes, reaches significantly control 1 The purpose of patients with type Ⅰ DM.Control group therapeutic effect is bad.
The blood glucose test results of the diabetic mice of 4. 33 Peptide C PU-PDLP1 immunization therapies STZ inductions of table
P compared with IA(5)-P2-1 ***’P ＜ 0.001；**’P ＜ 0.01；*P ＜ 0.05
The blood glucose of table 5.CPU-PDLP1 immune protection NOD mouse, morbidity and mortality result
The blood glucose of table 6.CPU-PDLP1 immunization therapy NOD mouse, morbidity and mortality result
Embodiment 3-6. be CPU-PDLP1 immunization therapies STZ of the present invention induction diabetes C57BL/6J heros mouse model with The detection of serum GLP-1 after prevention NOD mouse type 1 diabetes models：
The horizontal assay kits of GLP-1 are purchased from Mei Mian biotech firms in mice serum, and assay method illustrates according to kit It carries out.Brief experimental procedure is 1,50 μ l standard items or 5 times of diluted serum is added per hole.2,100 μ l enzyme marks are added per hole Secondary antibody, 37 DEG C of incubation 60min after preservative film winding.3, wash solution expire hole board-washing 4 times.4,50 μ l substrate As are added per hole With 50 μ l substrate Bs, gently mixing, 37 DEG C are protected from light and are incubated 15min.5,50 μ l terminate liquids (2M H are added per hole2SO4) (color is by indigo plant Turn yellow) 6, OD values are measured in 15min at 450nm wavelength.After C57BL/6J mouse immunes 7 times, GLP-1 water in serum is measured It is flat.Experimental result is as shown in table 7.The result shows that after immune CPU-PDLP1, in Mice Body, GLP-1 is horizontal significantly increases and molten Agent group is compared with LP1 with significant difference (P ＜ 0.01).
The testing result of serum GLP-1 after mouse is immunized in table 7.CPU-PDLP1
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；#P ＜ 0.05,##P ＜ 0.01；
Embodiment 3-7. be CPU-PDLP1 immunization therapies STZ of the present invention induction diabetes C57BL/6J heros mouse model with Serum insulin detects after preventing NOD mouse type 1 diabetes models：
Mice serum insulin level measurement is carried out according to kit specification.Brief experimental procedure is 1, standard items and sample The dilution of product and sample-adding.50 μ l of standard items or 5 times of diluted serum are accurately added on 96 orifice plates.2, it incubates, with sealing plate film Sealing plate is placed on 37 DEG C and incubates 30 minutes.3, it washs, is dried after discarding liquid, cleaning solution is filled it up with per hole, discarded after standing 30s, It is so repeated 5 times, pats dry.4,50 μ l of enzyme marking reagent are added per hole, except blank well.5, it incubates, operation is the same as 2.6, it washs, operation With 3.7, it develops the color, 50 μ l substrate As and 50 μ l substrate Bs is added per hole, gently mixing, 37 DEG C are protected from light incubation 15min.8, it terminates, often 50 μ l terminate liquids (2M H are added in hole2SO4) (color is turned yellow by indigo plant).9, it measures, blank well zeroing, each hole of 450nm wavelength measurements Absorbance (OD values).Shown in experimental result table 8.The result shows that CPU-PDLP1 is remarkably improved the pancreas islet in diabetic mice body It is plain horizontal, with solvent group and LP1 compared with statistical significance (**P ＜ 0.01,#P ＜ 0.05).
The testing result of serum insulin after mouse is immunized in table 8.CPU-PDLP1
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；#P ＜ 0.05,##P ＜ 0.01；
Embodiment 3-8. be CPU-PDLP1 immunization therapies STZ of the present invention induction diabetes C57BL/6J heros mouse model with Cytokines measurement after prevention NOD mouse type 1 diabetes models：
Cytokine assay is carried out according to kit specification.Brief experimental procedure be 1, standard items and sample dilution with Sample-adding.50 μ l of standard items or 5 times of diluted serum are accurately added on 96 orifice plates.2, it incubates, is placed on sealing plate film sealing plate 37 DEG C incubate 30 minutes.3, it washs, is dried after discarding liquid, fill it up with cleaning solution per hole, discarded after standing 30s, so repeatedly 5 It is secondary, it pats dry.4,50 μ l of enzyme marking reagent are added per hole, except blank well.5, it incubates, operation is the same as 2.6, it washs, operation is the same as 3.7, it shows Color 50 μ l substrate As and 50 μ l substrate Bs is added per hole, gently mixing, and 37 DEG C are protected from light incubation 15min.8, it terminates, 50 μ is added per hole L terminate liquids (2M H2SO4) (color is turned yellow by indigo plant).9, it measures, blank well zeroing, the absorbance (OD in each hole of 450nm wavelength measurements Value).Experimental result is shown in Table 9, the results showed that, after C57BL/6J and NOD mouse are immunized in CPU-PDLP1, IL-10 is horizontal significantly to be risen Height, IFN-γ level significantly reduce, and illustrate exempting from for Th2 mediations with statistical significance (P ＜ 0.05) compared with solvent group and LP1 Epidemic disease response increases, and the immune response that Th1 is mediated declines.This proves that CPU-PDLP1 can adjust Th1/Th2 in diabetic mice body It is unbalance, it is more to intervene immunologic balance to Th2 conversions.CPU-PDLP1 it is immune can be used for treat drawn by internal Th1/Th2 is unbalance The disease risen, such as type 1 diabetes and its complication, relevant disease that other Th1/Th2 such as inflammatory reaction are unbalance.
Serum cytokines testing result after STZ diabetic mices is immunized in table 9.CPU-PDLP1
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；#P ＜ 0.05,##P ＜ 0.01；
Serum cytokines testing result after table 10.CPU-PDLP1 prevention NOD diabetic mices
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；#P ＜ 0.05,##P ＜ 0.01；
Embodiment 3-9. is the diabetes C57BL/6J heros mouse model of CPU-PDLP immunization therapies STZ of the present invention inductions and prevents Control serum lipids, uric acid, creatinine, xanthine oxidase (XOD) and anti-oxidation stress index after NOD mouse type 1 diabetes models Detection；
The detection reagent of mice serum blood fat, uric acid, xanthine oxidase (XOD) and anti-oxidation stress is carried by building up company For being detected and calculating according to kit explanation.Experimental result is as shown in table 11 and table 12, the results showed that CPU-PDLP1 exempts from After epidemic disease mouse, serum creatinine and uric acid and level are remarkably decreased, and have significant difference (P ＜ compared with solvent group and LP1 0.01).Antioxidation significantly increases, and has significant difference (P ＜ 0.05).Blood fat significantly reduces, and has significant difference (P ＜ 0.05).Therefore diabetic complication can be alleviated, protect kidney.
Serum lipids, uric acid, creatinine, xanthine oxidase (XOD) after STZ diabetic mices is immunized in table 11.CPU-PDLP1 Testing result
P compared with IA(5)-P2-1 *P ＜ 0.05,**P ＜ 0.01；#P ＜ 0.05,##P ＜ 0.01；
Table 12.CPU-PDLP1 be immunized serum oxidative after STZ diabetic mices stress index testing result
P compared with IA(5)-P2-1 **P ＜ 0.01*P ＜ 0.05；0.05 #P ＜ of@P ＜, 0.05 ＆P ＜ 0.05
Embodiment 3-10. be CPU-PDLP1 immunization therapies STZ of the present invention induction diabetes C57BL/6J heros mouse model with Pancreas inflammatory pathological analysis is identified after treating NOD mouse type 1 diabetes；
At the end of zoopery, take experimental group and control animals tissue as fresh as possible, PBS (phosphate buffer) It washes, takes and fix and embed less than 0.5cm × 0.5cm × 0.1cm tissue blocks, be sliced, Hematoxylin-eosin (HE) dyeing.HE is dyed Steps are as follows：Hematoxylin dyes 3min, and 1% hydrochloride alcohol breaks up 30s, eosin stains, through concentration from low to high after pure water rinsing Dehydration of alcohol, dimethylbenzene is transparent, after neutral gum mounting light under the microscope, carry out 200 × microphotograph.Experimental result is as schemed Shown in 2, the experimental results showed that, after CPU-PDLP1 (Fig. 2A, Fig. 2 B) immunization therapy mouse, with solvent and P277 or LP1 to photograph Than mice pancreatic inflammation significantly mitigates, and pancreas is complete, is clearly outlined, and can develop protection pancreas drug, treat diabetes B.
Embodiment 3-11. is diabetes C57BL/6J heros mouse and the prevention of wood invention CPU-PDLP1 immunization therapies STZ inductions The insulin of pancreas, glucagon, CD4+T cells and CD8+T cellular immunity groupizations mirror after NOD mouse type 1 diabetes models It is fixed；
At the end of zoopery, take experimental group and control animals tissue as fresh as possible, PBS (phosphate buffer) It washes, takes and fix and embed less than 0.5cm × 0.5cm × 0.1cm tissue blocks, be sliced, insulin antibody processing, drop are added dropwise on slice Add biotinylated secondary antibody (IgG) to handle, then horseradish enzyme label strepto- avidin working solution (S-A/HRP) is added dropwise, 37 DEG C 20 Minute, 0.1MPBS is washed 3 times × 5 minutes；, DAB colour developings, washing, resinene mounting, micro- sem observation：Choice experiment group and right According to group the positive and negative tissue carry out 200 × microphotograph, software analyze sxemiquantitative.Experimental result is as shown in figure 3, experiment The result shows that after the diabetes C57BL/6J hero mouse of CPU-PDLP1 (Fig. 3) immunization therapies STZ inductions, with solvent and P277 groups pair Photograph ratio, pancreatic insulin secretion dramatically increase, and Fig. 4 results are shown：CPU-PDLP1 prevents NOD mouse type 1 diabetes models Afterwards, compared with solvent and P277 groups compare, mice pancreatic significantly improves, and pancreas is complete, sharp outline, islet secretion pancreas islet Element amount obviously increases (Fig. 4 A), and islet secretion glucagon secretion significantly reduces (Fig. 4 B), and CD4+T cells dramatically increase (figure 5A), CD8+T cells significantly reduce (Fig. 5 B), can develop protection and adjust pancreatic secretion function medicament.
Embodiment 4-1-11. is spleen cell after CPU-PDLP1 immune protections NOD mouse type 1 diabetes models of the present invention Th1, Th2, Th17, Treg content analysis are identified；
At the end of zoopery, experimental group and the fresh spleen of control animals tissue, conventional treatment is taken to prepare spleen cell, It is marked, washs according to flow cytometer showed conventional method, detection Th1 (Fig. 6 A), Th2 (Fig. 6 B), Th17 (Fig. 6 C), Treg (figures 6D) measure.As a result as Fig. 6 is shown：Th2 and Treg cell concentrations dramatically increase after NOD mouse are immunized in CPU-PDLP1, and Th1, Th17 are aobvious Writing reduces.
Embodiment 4-1-12. is HbAle after CPU-PDLP1 immune protections NOD mouse type 1 diabetes models of the present invention Protein Detection
The horizontal detection of glycosylated hemoglobin (glycosylated hemoglobin, GHb) is using saccharification blood in mice serum Lactoferrin (GHb) test kit is measured (Bioengineering Research Institute is built up in Nanjing).Operating process is as follows：1. red thin Born of the same parents wash：It takes EDTA or anticoagulant heparin whole blood 2-4ml to be placed in centrifuge tube with a scale, 10min is centrifuged with 800rpm, is discarded supernatant Red blood cell is collected, is then washed as stated above with physiological saline 2 times.2. the preparation of hemolysate：Precipitation red blood cell 1ml is taken to be added Distilled water 1.5ml shakes 3min with hand fierceness and hemolysate is made, 10 μ l reagent addings of hemolysate, four 2.5ml, mixing, room temperature is taken to put 10min is set, with spectrophotometer at 540nm, each pipe absorbance is surveyed in the zeroing of 1cm optical path water, by gained absorbance × 0.3677 The as content of Hb (g/ml).Calculation formula is as follows：Per 10g hemoglobin absorptions degree=(measuring pipe OD values-blank tube OD values) * 10/ (hemoglobin grams/milliliter hemolysate), is shown in Fig. 7, as a result shows：26-38 weeks CPU-PDLP1 (100 μ g) administration group mouse Blood glucose is controlled effectively, and mouse glycosylated hemoglobin is 4.45%, reaches the normal water of American Diabetes Association ADA requirements It is flat.
Embodiment 4-1-13. is resistance to for sugar after 33 Peptide C PU-PDLP1 immune protection NOD mouse type 1 diabetes models of the invention It is fixed to measure
After being immunized at the 7th time, the glucose tolerance situation of administration group and control group mice is measured.Every group takes 3 mouse, often The blood glucose value of mouse is near this group of average blood sugar value.After mouse is deprived of food but not water 16h, every mouse peritoneal injection dosage is The glucose solution of 2mg/kg.The mouse injection dosage of a concentration of 400mg/ml of glucose solution, weight 20g are 0.1ml.Note 0min, 15min, 30min after glucose are penetrated, 60min, 120min measure the blood glucose value of mouse in such a way that tail vein takes blood. And blood is taken in 15min after having injected glucose, it measures serum insulin level result and sees that Fig. 8, Peptide C PU-PDLP1 immune groups are small Mouse has preferable glucose tolerance, and 120min after glucose is injected intraperitoneally, and CPU-PDLP1 group mouse blood sugar values are restored to just Ordinary water is flat.Area is significantly less than other groups (* * P ＜ 0.01 under the sugar tolerance empirical curve of CPU-PDLP1 group mouse；##P ＜ 0.05) Peptide C PU-PDLP1 effectively improves the glucose-tolerant situation of diabetic mice, improves the glycometabolism in Mice Body.
Conclusion：The 33 Peptide C PU-PDLP1 of the present invention can induce the Th2 antibody of the specifically B cell epitope of targeting DPP4 (Th1 will produce IgG2a subclass antibodies in Mice Body, and Th2 types then will produce IgG1 subclass antibodies, also will produce spy in mankind's body Different subclass antibodies), the generation that Th2 subclass antibodies can increase Th2 type cytokines adjusts the immune rebalancing for tending to Th2 and controls The higher diseases of Th1 processed alleviate symptom, and generate the antibody of the energy and DPP4 specific bindings of higher level, increase GLP-1, pancreas Island element, raises IL-10, lowers IFN-γ, increases Th2 and Treg cell concentrations, reduces Th1 and Th17 cell concentrations, mitigates pancreatitis Disease protects islet β cell insulin function, lowers pancreas islet glucagon secretion, reduces blood glucose and uric acid and creatinine, sweet Oily three esters, XOD enhance anti-oxidative stress, and potency reaches 20000 after animal is immunized in 33 Peptide C PU-PDLP1, can be used for making Standby antibody.The coupling protein that is prepared based on 33 Peptide C PU-PDLP1 and its nucleic acid sequence, polymer, composition or using it as raw material What is prepared is used for DPP4 exceptions, uric acid creatinine exception, pathoglycemia, oxidative stress exception, dysglycemia and panimmunity Abnormal balance includes Th1/Th2 unbalance, CD4+/CD8+ exceptions, Th17 and the extremely caused diabetes of Treg, nephrosis, artery congee Drug on the higher treating correlative diseases of the Th1 such as sample hardening, senile dementia, eye disease, pedopathy, angiocarpy is widely used.
Finally illustrate, 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 in the form and details make it various changes, defined by the appended claims The spirit and scope of the present invention.

Claims (10)

1. a linear 33 peptide molecule CPU-PDLP1, the amino acid sequence is shown in SEQ ID No.1, by people's dipeptidyl peptidase 4 (DPP4) the 23 peptide LP1 compositions of a B cell epitope D1 and an epitope containing Th2, the B cell antigen epi-position D1 of people DPP4 There is peptide VFLENSTFDE amino acid sequences composition, the amino acid sequence to see SEQ ID No.1.
2. 23 peptide LP1 of an epitope containing Th2 according to claim 1, the amino acid sequence are shown in SEQ ID No.2.
3. 23 peptide LP1 of an epitope containing Th2 according to claim 2, from 1 Th2 cell epitopes transformation, this Th2 cell epitopes derive from 1 of the N-terminal of 1 P277 peptide of upper 437~460 of human heat shock protein HSP60 by 13 amino The Th2 epitope P2 of acid composition form LP1 as soon as connecting section connection peptide being made of 10 amino acid residues in its N-terminal.
4. the amino acid sequence of the connection peptide L1 of 10 amino acid residues composition according to claim 3 is： GFEYQDSGTI, the amino acid sequence are shown in SEQ ID No.3, wherein deriving from human pancreatic island cell specific antigen-containing one A B cell epitope of (IA-2) membrane-proximal region of insulinoma GAP-associated protein GAP -2 JM2, amino acid sequence are：FEYQD is named as IA2 (5) adds amino acid Gly in its N-terminal, in C-terminal plus the peptide SGTI of 4 amino acid sequences, just constitutes L1.
5. the various peptide molecules and its coupling protein and fusion protein according to claim 1-4 can be used for recombinating micro- life Object and transgenic animals or plant and its cell make them as production plant, can produce peptide based immunogens and egg containing 33 peptides White immunogene and its fusion protein, coupling protein or series and parallel object are made oral vaccine and are produced based on above-mentioned peptide or protein Antibody special DPP4.
6. various peptides described in the claim 1-5 comprising immunological effective amount and combinations thereof or fusion protein, coupling egg White or series and parallel object.
7. the composition or fusion protein, coupling protein according to claim 5-6 or series and parallel object, it is characterised in that：Also Including pharmaceutically acceptable carrier and pharmaceutically acceptable auxiliary material, carrier and pharmaceutically active substance.
8. the effect of the various peptides according to claim 1-7, connection peptide L1 constitutes peptide LP1, LP1 again for connecting T epitopes The immunogenicity of B epitope can be significantly increased by being connected with B epitope, generate the antibody of high titre, and exploitation antibody can treat disease Specific antigen related to detection DPP4.
9.33 peptides and albumen and peptide or polymer are prepared based on it, immune animal can generate the energy and DPP4 specificity of high titre In conjunction with Th2 antibody, this antibody can inhibit DPP4 activity and adjust the immune rebalancing for tending to Th2, per being given with this 33 peptide within 2-3 weeks Medicine can prevent and treat type 1 diabetes NOD mouse and treat the diabetic mice of STZ inductions, significantly can control or reverse blood Sugar, NOD mouse blood sugars are normal, GLP-1 and Th2 cell factors significantly increase, and Th1 cell factors significantly reduce, and significantly change It has been apt to incidence, the death rate, panimmunity balance and uric acid, creatinine and blood fat and oxidative stress target improvement, pancreas inflammatory to subtract Gently, pancreatic secretion insulin ability significantly increases, and glucagon significantly reduces.
10.33 peptides and based on 33 peptides include with microorganism, animal and plant and cell expression prepare vaccine and drug, preventing With treatment diabetes especially type 1 diabetes and DPP4 exceptions, pathoglycemia, Th1 is higher, CD4+/CD8+ is abnormal, Th17 and Treg is abnormal, oxidative stress is abnormal, complication caused by uric acid creatinine exception and dysglycemia includes that nephrosis, artery are athero- Have in the disease higher Th1 such as hardening, uric acid creatinine blood fat oxidative stress exception, senile dementia, eye disease, pedopathy, angiocarpy Important value.
CN201810434955.6A 2018-05-03 2018-05-03 It is a kind of to prevent and treat type 1 diabetes and with linear 33 peptide and application thereof of protection Pancreatic beta cells function Pending CN108586619A (en)

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